Xylanases for solubilising arabinoxylan-containing material

ABSTRACT

The present invention relates to a method for solubilising arabinoxylan-containing material (particularly insoluble arabinoxylan-containing material), comprising admixing a xylan-containing material with a xylanase comprising a polypeptide sequence shown herein as SEQ ID No. 3, SEQ ID No. 2, SEQ ID No. 1, SEQ ID No. 9, SEQ ID No. 10, SEQ ID No. 11 or SEQ ID No. 15, or a variant, homologue, fragment or derivative thereof having at least 75% identity with SEQ ID No. 3 or SEQ ID No. 2 or SEQ ID No. 1 or SEQ ID No. 9 or SEQ ID No. 10 or SEQ ID No. 11 or SEQ ID No. 15; or a polypeptide sequence which comprises SEQ ID No. 3. SEQ ID No. 2, SEQ ID No. 1, SEQ ID No. 9, SEQ ID No. 10, SEQ ID No. 11 or SEQ ID No. 15 with a conservative substitution of at least one of the amino acids; or a xylanase which is encoded by a nucleotide sequence shown herein as SEQ ID No. 6, SEQ ID No. 5, SEQ ID No. 4, SEQ ID No. 12, SEQ ID No. 13, SEQ ID No. 14, SEQ ID No. 16, SEQ ID No. 17 or SEQ ID No. 18, or a nucleotide sequence which can hybridize to SEQ ID No. 6, SEQ ID No. 5, SEQ ID No. 4, SEQ ID No. 12, SEQ ID No. 13, SEQ ID No. 14, SEQ ID No. 16, SEQ ID No. 17 or SEQ ID No. 18 under high stringency conditions, or a nucleotide sequence which has at least 75% identity with SEQ ID No. 6, SEQ ID No. 5, SEQ ID No. 4, SEQ ID No. 12, SEQ ID No. 13, SEQ ID No. 14, SEQ ID No. 16, SEQ ID No. 17 or SEQ ID No. 18, or a nucleotide sequence which differs from SEQ ID No. 6 or SEQ ID No. 5 or SEQ ID No. 4 or SEQ ID No. 12 or SEQ ID No. 13 or SEQ ID No. 14 or SEQ ID No. 16 or SEQ ID No. 17 or SEQ ID No. 18 due to the degeneracy of the genetic code, or a xylanase obtainable (or obtained) from  Fusarium verticilloides.  The present invention also relates to a novel xylanase comprising (or consisting of) a polypeptide sequence shown herein as SEQ ID No. 3, SEQ ID No. 2 or SEQ ID No. 1, or a variant, homologue, fragment or derivative thereof having at least 99% identity with SEQ ID No. 3 or SEQ ID No. 2 or SEQ ID No. 1; or a xylanase which is encoded by a nucleotide sequence shown herein as SEQ ID No. 6, SEQ ID No. 5 or SEQ ID No. 4, or a nucleotide sequence which can hybridize to SEQ ID No. 4 or SEQ ID No. 5 under high stringency conditions, or a nucleotide sequence which has at least 97.7% identity (preferably 98% identity) with SEQ ID No. 6, SEQ ID No. 5 or SEQ ID No. 4. The present invention yet further relates to methods relating to feedstuffs, malting and brewing, processing of grain-based materials such as during the production of bioethanol or biochemical (e.g. bio-based isopropanol), or wheat gluten-starch separation processes and the like.

FIELD OF THE INVENTION

The present invention relates to the use of xylanases having unusualproperties in applications, including in feedstuffs, in brewing ormalting, in the treatment of arabinoxylan containing raw materials likegrain-based materials, e.g. in the production of biofuel or otherfermentation products, including biochemicals (e.g. bio-based isoprene),and/or in the wheat gluten-starch separation industry, and methods usingthese xylanases, as well as compositions (such as feed additivecompositions) comprising said xylanases. The present invention alsorelates to a new xylanase with unusual properties that renders it usefulin applications, including in feedstuffs, in brewing or malting, in thetreatment of arabinoxylan containing raw materials like grain-basedmaterials, e.g. in the production of biofuel or biochemicals (e.g.bio-based isoprene), and/or in the wheat gluten-starch separationindustry.

BACKGROUND OF THE INVENTION

For many years, endo-β-1,4-xylanases (EC 3.2.1.8) (referred to herein asxylanases) have been used for the modification of complex carbohydratesderived from plant cell wall material. It is well known in the art thatthe functionality of different xylanases (derived from differentmicroorganisms or plants) differs enormously. Xylanase is the name givento a class of enzymes which degrade the linear polysaccharidebeta-1,4-xylan into xylooligosaccharides or xylose, thus breaking downhemicellulose, one of the major components of plant cell walls.

Based on structural and genetic information, xylanases have beenclassified into different Glycoside Hydrolase (GH) families (Henrissat,(1991) Biochem. J. 280, 309-316).

Initially all known and characterized xylanases belonged to the familiesGH10 or GH11. Further work then identified numerous other types ofxylanases belonging to the families, GH5, GH7, GH8 and GH43 (Collins etal (2005) FEMS Microbiol Rev., 29 (1), 3-23).

Until now the GH11 family differs from all other GH's, being the onlyfamily solely consisting of xylan specific xylanases. The structure ofthe GH11 xylanases can be described as a β-Jelly roll structure or anall β-strand sandwich fold structure (Himmel et al 1997 Appl. Biochem.Biotechnol. 63-65, 315-325). GH11 enzymes have a catalytic domain ofaround 20 kDa.

GH10 xylanases have a catalytic domain with molecular weights in therange of 32-39 kDa. The structure of the catalytic domain of GH10xylanases consists of an eightfold ⊕/α barrel (Harris et al 1996—Acta.Crystallog. Sec. D 52, 393-401).

Three-dimensional structures are available for a large number of FamilyGH10 enzymes, the first solved being those of the Streptomyces lividansxylanase A (Derewenda et al J Biol Chem 1994 Aug. 19; 269(33) 20811-4),the C. fimi endo-glycanase Cex (White et al Biochemistry 1994 Oct. 25;33(42) 12546-52), and the Cellvibrio japonicus Xyn10A (previouslyPseudomonas fluorescens subsp. xylanase A) (Harris et al Structure 1994Nov. 15; 2(11) 1107-16.). As members of Clan GHA they have a classical(α/β)_(ϵ)TIM barrel fold with the two key active site glutamic acidslocated at the C-terminal ends of beta-strands 4 (acid/base) and 7(nucleophile) (Henrissat et al Proc Natl Acad Sci USA 1995 Jul. 18;92(15) 7090-4).

Comprehensive studies characterising the functionality of xylanases havebeen done on well characterised and pure substrates (Kormelink et al.,1992 Characterisation and mode of action of xylanases and some accessoryenzymes. Ph.D. Thesis, Agricultural University Wageningen, Holland (175pp., English and Dutch summaries)). These studies show that differentxylanases have different specific requirements with respect tosubstitution of the xylose backbone of the arabinoxylan (AX). Somexylanases require three un-substituted xylose residues to hydrolyse thexylose backbone; others require only one or two. The reasons for thesedifferences in specificity are thought to be due to the threedimensional structure within the catalytic domains, which in turn isdependent on the primary structure of the xylanase, i.e. the amino acidsequence. However, the translation of these differences in the aminoacid sequences into differences in the functionality of the xylanases,has up until now not been documented when the xylanase acts in a complexenvironment, such as a plant material, e.g. in a feedstuff.

The xylanase substrates in plant material, e.g. in wheat, havetraditionally been divided into two fractions: The water un-extractableAX (WU-AX) and the water extractable AX (WE-AX), There have beennumerous explanations as to why there are two different fractions of AX.The older literature (D'Appolonia and MacArthur—(1976, Cereal Chem. 53.711-718) and Montgomery and Smith (1955, J. Am. Chem. Soc. 77. 3325-332)describes quite high differences in the substitution degree betweenWE-AX and WU-AX. The highest degree of substitution was found in WE-AX.This was used to explain why some of the AX was extractable. The highdegree of substitution made the polymer soluble, compared to a lowersubstitution degree, which would cause hydrogen bonding between polymersand consequently precipitation.

The difference between the functionality of different xylanases has beenthought to be due to differences in xylanase specificity and therebytheir preference for the WU-AX or the WE-AX substrates.

Xylanase enzymes have been reported from nearly 100 different organisms,including plants, fungi and bacteria. The xylanase enzymes areclassified into several of the more than 40 families of glycosylhydrolase enzymes. The glycosyl hydrolase enzymes, which includexylanases, mannanases, amylases, β-glucanases, cellulases, and othercarbohydrases, are classified based on such properties as the sequenceof amino acids, their three dimensional structure and the geometry oftheir catalytic site (Gilkes, et al., 1991, Microbiol. Reviews 55:303-315).

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows a polypeptide sequence (SEQ ID No. 1) of a xylanase of thepresent invention (FveXyn4). This is the pre-pro-protein. Underlined(lower case) portion of the sequence reflects an N terminal signalpeptide can be cleaved before the enzyme is matured. The amino acidsshown in bold and italicized may also be cleaved by post-translationalmodification before the enzyme is fully matured.

FIG. 2 shows a polypeptide sequence (SEQ ID No. 2) of a xylanase of thepresent invention (FveXyn4). This is the pro-protein. The amino acidsshown in bold and italicized may also be cleaved by post-translationalmodification before the enzyme is fully matured.

FIG. 3 shows a polypeptide sequence (SEQ ID No. 3) of a xylanase of thepresent invention (FveXyn4). This is the active form of the enzyme. Thismay be referred to herein as the mature form of the enzyme.

FIG. 4 shows a nucleotide sequence (SEQ ID No. 4) encoding a xylanase ofthe present invention (FveXyn4). The lower case nucleotides which are inbold show the intron sequence. The signal sequence is shown bold (uppercase).

FIG. 5 shows a nucleotide sequence (SEQ ID No. 5) encoding a xylanase ofthe present invention (FveXyn4). The signal sequence is shown bold(uppercase).

FIG. 6 shows a nucleotide sequence (SEQ ID No. 6) encoding a xylanase ofthe present invention (FveXyn4).

FIG. 7 shows a scheme of an auto-analyzer for the determination ofpentosan by an automated phloroglucinol method: (a) Acetic acid mixedwith HCl; (b) air bubbling; (c) phloroglucinol in ethanol; (d) sample;(e) sample accelerator; (f) flow cell way-out; (g) peristaltic pump; (h)glass coil; (i) thermostat (96° C.); (j) multiple wavelengthspectrophotometer (410, 510, 550, and 620 nm); (k) waste; (l) computer(Rouau & Surget, 1994 Carbohydrate Polymers, 24, 123-32).

FIG. 8 shows pentosan (C-5 sugar) release (solubilisation of pentosans)from wheat bran as a function of xylanase dosage. The xylanases usedwere the xylanase of the present invention (FveXyn4) compared with abenchmark xylanase namely Econase® XT.

FIG. 9 shows solubilisation of pentosans from cDDGS as a function ofxylanase dosage. The xylanases used were the xylanase of the presentinvention (FveXyn4) compared with a benchmark xylanase namely Econase®XT.

FIG. 10 shows a plasmid map of pZZH254.

FIG. 11 shows the pH activity profile of FveXyn4.

FIG. 12 shows the temperature profile of FveXyn4.

FIG. 13 shows viscosity reduction on high viscosity wheat by FveXyn4 inan in vitro animal model (where pH was maintained at 6.0).

FIG. 14 shows RVA viscosity profiles in whole ground wheat starting atpH 5.2 for the xylanase in accordance with the present invention namelyFveXyn4 and the benchmark enzymes Xylathin™.

FIG. 15 shows a polypeptide sequence (SEQ ID No. 9) of a xylanase of thepresent invention (FoxXyn2). This is the pre-pro-protein. Underlined(lower case) portion of the sequence may reflect an N terminal signalpeptide which can be cleaved before the enzyme is matured. The aminoacids shown in bold and italicized may also be cleaved bypost-translational modification before the enzyme is fully matured.

FIG. 16 shows a polypeptide sequence (SEQ ID No. 10) of a xylanase ofthe present invention (FoxXyn2). This is the pro-protein. The aminoacids shown in bold and italicized may also be cleaved bypost-translational modification before the enzyme is fully matured. Thissequence may be an active form of the protein and may be one active formof the protein. This may be referred to herein as the mature form of theenzyme.

FIG. 17 shows a polypeptide sequence (SEQ ID No. 11) of a xylanase ofthe present invention (FoxXyn2). This is another active form of theenzyme, in some embodiments, this may be referred to herein as themature form of the enzyme.

FIG. 18 shows RVA viscosity profiles in whole ground wheat starting atpH 5.2 for the xylanase in accordance with the present invention namelyFoxXyn2 and the benchmark enzymes Xylathin™.

FIG. 19 shows a nucleotide sequence (SEQ ID No. 12) encoding a xylanaseof the present invention (FoxXyn2). The lower case nucleotides which arein bold show the intron sequence. The signal sequence is shown bold(upper case).

FIG. 20 shows a nucleotide sequence (SEQ ID No. 13) encoding a xylanaseof the present invention (FoxXyn2). The signal sequence is shown bold(upper case).

FIG. 21 shows a nucleotide sequence (SEQ ID No. 14) encoding a xylanaseof the present invention (FoxXyn2).

FIG. 22 shows a plasmid map of pZZH135.

FIG. 23 shows the pH profile of FoxXyn2.

FIG. 24 shows the temperature profile of FoxXyn2.

FIG. 25 shows a polypeptide sequence (SEQ ID No. 15) of a xylanase ofthe present invention (from Fusarium)—Fusarium Comparative SequencingProject, Broad Institute of Harvard and MIT(http://www.broadinstitute.org/)). In some embodiments, this may bereferred to herein as the mature form of the enzyme.

FIG. 26 shows an alignment of the mature proteins for FveXyn4 (SEQ IDNo. 3), FoxXyn2 (SEQ ID No. 11) and the xylanase shown herein as SEQ IDNo. 15.

FIG. 27 shows the effect of the xylanase and protease treatments aloneand in combination on the solubilization of pentosan and protein frominsoluble corn DDGS. Letters a-d are significant different according toon-way ANOVA and Holm-Sidak comparisons with overall significance levelat P=0.05. Error bars indicate S.D.

FIG. 28 shows the effect of the xylanase and protease treatments aloneand in combination on the solubilization of pentosan and protein frominsoluble wheat DDGS. Letters a-d are significant different according toon-way ANOVA and Holm-Sidak comparisons with overall significance levelat P=0.05. Error bars indicate S.D.

FIG. 29 shows solubilisation of pentosans from cDDGS as a function ofxylanase dosage. The xylanases used were the xylanases of the presentinvention (FveXyn4 and FoxXyn2).

FIG. 30 shows a nucleotide sequence (SEQ ID No. 16) encoding a xylanasefor use in the present invention from Fusarium—obtained from FusariumComparative Sequencing Project, Broad Institute of Harvard and MIT(http://www.broadinstitute.org/)). The lower case nucleotides which arein bold show the intron sequence. The signal sequence is shown bold(uppercase). Changes compared with SEQ ID No. 4 are underlined.

FIG. 31 shows a nucleotide sequence (SEQ ID No. 17) encoding a xylanasefor use in the present invention from Fusarium—obtained from FusariumComparative Sequencing Project, Broad Institute of Harvard and MIT(http://www.broadinstitute.org/)). The signal sequence is shown bold(upper case). Changes compared with SEQ ID No. 5 are underlined.

FIG. 32 shows a nucleotide sequence (SEQ ID No. 18) encoding a xylanasefor use in the present invention from Fusarium—obtained from FusariumComparative Sequencing Project, Broad Institute of Harvard and MIT(http://www.broadinstitute.org/)). Changes compared with SEQ ID No. 6are underlined.

SUMMARY OF THE INVENTION

A seminal finding of the present invention is a novel xylanase isolatedfrom Fusarium verticilloides, which enzyme has surprising and unexpectedproperties. In particular it is unexpectedly good at breaking down(solubilising) insoluble arabinoxylans (AXinsol). Surprisingly theenzyme has been found to efficiently breakdown (solubilise) AXinsol froma wide range of substrates, including corn, wheat, DDGS, etc, inparticular corn and corn based substrates, in particular both wheat(including wheat-based) products and corn (including corn-basedproducts). This contrasts with prior-known enzymes, which are ofteninferior at solubilising AXinsol in corn or corn-based substrates orwhich are not efficient in both wheat- and corn-based substrates.

In addition, the enzyme of the present invention is particularly good atnot only breaking down (solubilising) AXinsol, but also breaking down(or degrading) the solubilized polymers efficiently. By being able toefficiently (quickly) breakdown (degrade) the solubilized polymers(obtained from dissolving AXinsol), a (fast) reduction in viscosity isobtained or the solubilized polymers (obtained from dissolving AXinsol)cannot contribute to increasing viscosity. This latter effect isessential in some of the claimed applications.

Without wishing to be bound by theory, the enzyme of the presentinvention mainly releases polymers, which do not contribute toviscosity, because the released polymers are short.

For the first time, the present inventors have isolated and sequencedthis novel xylanase.

Typically, conventional xylanases may breakdown AXinsol, but will oftenlead to an increase in viscosity of the mixture. This increasedviscosity is disadvantageous in many applications.

Without wishing to be bound by theory, although some conventionalxylanases breakdown AXinsol, they lead to an increase in solubledegradation products of high molecular weight, which leads to anincrease in viscosity in the mixture.

Furthermore or alternatively and again without wishing to be bound bytheory, conventional xylanase enzymes may breakdown AXinsol, but becausethey do not degrade the solubilised products of high molecular weightfast enough the viscosity in the mixture is not ideal. In contrast, withthe methods and uses of the present invention, the xylanases breakdownAXinsol without increasing viscosity and/or whilst reducing viscosityquickly compared with conventional enzymes. Without wishing to be beingbound by theory, it is believed that high molecular weight products arenot formed by the enzymes of the present invention.

The enzymes of the present invention and as described herein have beenfound to not only breakdown (solubilise) insoluble arabinoxylans(AXinsol) from a wide range of substrates, including corn, wheat, DDGS,etc, in particular corn and corn-based substrates, in particular bothwheat (including wheat-based) products and corn (including corn-basedproducts), but also efficiently ensuring that viscosity is not raisedand/or reducing viscosity. Without wishing to be being bound by theory,it is believed that high molecular weight products are not formed by theenzymes of the present invention.

Thus the present invention relates to enzymes capable of solubilisingpentosans, in particular xylan-containing materials, such asarabinoxylans, in particular insoluble arabinoxylans. In particular theenzyme is particularly good at solubilising pentosans in particularxylan-containing materials, such as arabinoxylans, in particularinsoluble arabinoxylans, in a broad spectrum of substrates, includingcorn based substrates.

The present invention further relates to enzymes capable of degradingAXsol or the breakdown products of AXinsol to ensure viscosity is notincreased and/or is reduced in the reaction mixture.

Many of the xylanases commercialized for use in feedstuffs forsolubilizing pentosans are GH11 enzymes. It had been considered by thoseskilled in the art that GH10 xylanases were not as strong at solublizingpentosans, particularly AXinsol, compared with GH11 xylanases.Surprisingly it has been found that the novel xylanase disclosed hereinwhich is a GH10 xylanase is particularly good at degrading AXinsol in abroad spectrum of substrates, including corn based substrates.Surprisingly, the present inventors have found that the GH10 xylanasesof the present invention outperform commercial GH11 xylanases in theirability to solubilize pentosans.

The fact that the present enzymes efficiently degrade AXinsol from cornand corn-based substrates is significantly advantageous as corn holdsmuch more AX in the insoluble form compared with other cereals, such aswheat and rye for example. Therefore only xylanases that can breakdownAXinsol can show significant benefit, to animals fed on corn-based diet,such as corn-soy diet for example.

It was completely unexpected for a GH10 xylanase to be so good ondegrading AXinsol in cereals, particularly in corn or corn-basedsubstrates.

The enzymes of the present invention are able to efficiently (andquickly) degrade the polymers and oligomers that are produced fromdegradation of AXinsol or that are present in grain-based material. Thisleads to an unexpected advantage for the GH10 xylanases taught herein inthat they are particularly good in a number of applications to keepviscosity low or to reduce viscosity, e.g. in feedstuffs; in brewingand/or malting; in grain-based production of glucose, e.g. for furtherprocessing to biofuels and/or biochemicals (e.g. bio-based isoprene); orin the wheat gluten-starch separation industry for the production ofstarch for example.

Notably it has been found that the degradation product on average isshorter for the GH10 enzymes tested herein compared with GH11 enzymes.This means that the degradation products do not contribute to or causean increase in viscosity.

Based on these findings, the xylanases according to the presentinvention can be used to degrade a xylan-containing material,particularly arabinoxylans, particularly AXinsol. In addition oralternatively, the xylanases according to the present invention can beused to degrade soluble polymers (e.g. oligomers) that are produced fromdegradation of AXinsol or that are (naturally) present in grain-basedmaterials. Surprisingly it has been found that the xylanases accordingthe present invention can be used to both degrade a xylan-containingmaterial, particularly arabinoxylans, particularly AXinsol, and todegrade soluble polymers (e.g. oligomers) that are produced fromdegradation of AXinsol.

Such enzymes finds useful application in many industries, includingfeedstuffs, malting and brewing, in the treatment of arabinoxylancontaining raw materials like grain-based materials, in the wheatgluten-starch separation industry, in the production of starch derivedsyrups, in biofuel production, and the like.

Only through the isolation and testing of FveXyn 4 (SEQ ID No. 3) werethese surprising and unexpected technical effects found. Thereafter itwas possible to identify other similar acting enzymes (e.g. SEQ ID No.11 and/or SEQ ID No. 15).

Statements of the Invention

According to a first aspect the present invention provides a xylanasecomprising (or consisting of) a polypeptide sequence shown herein as SEQID No. 1, SEQ ID No. 2 or SEQ ID No. 3, or a variant, homologue,fragment or derivative thereof having at least 98.5% (e.g. at least 98.8or 99 of 99.1 or 99.5%) identity with SEQ ID No. 1 or SEQ ID No. 2 orSEQ ID No. 3; or a xylanase which is encoded by a nucleotide sequenceshown herein as SEQ ID No. 4, SEQ ID No. 5 or SEQ ID No. 6, or anucleotide sequence which has at least 97.7% (e.g. at least 98%, 98.5%or 99%) identity with SEQ ID No. 4, SEQ ID No. 5 or SEQ ID No. 6.

In a further aspect the present invention provides an isolated orrecombinant nucleic acid molecule comprising (or consisting of) apolynucleotide sequence selected from the group consisting of:

-   -   a. a polynucleotide sequence which encodes a polypeptide        sequence selected from the group consisting of SEQ ID No. 1, SEQ        ID No. 2 or SEQ ID No. 3, or a variant, homologue, fragment or        derivative thereof having at least 98.5% (e.g. at least 98.8 or        99 or 99.1 or 99.5%) identity with SEQ ID No. 1 or SEQ ID No. 2        or SEQ ID No. 3; or    -   b. a polynucleotide sequence shown herein as SEQ ID No. 4, SEQ        ID No. 5 or SEQ ID No. 6; or a nucleotide sequence which has at        least 97.7% (e.g. at least 98%, 98.5% or 99%) identity with SEQ        ID No. 4, SEQ ID No. 5 or SEQ ID No. 6.

In one aspect of the present invention there is provided a vector (e.g.a plasmid) comprising a polynucleotide sequence selected from the groupconsisting of:

-   -   a. a polynucleotide sequence which encodes a polypeptide        sequence selected from the group consisting of SEQ ID No. 1, SEQ        ID No. 2 or SEQ ID No. 3, or a variant, homologue, fragment or        derivative thereof having at least 98.4% (e.g. at least 98.5% or        98.8 or 99 or 99.1 or 99.5%) identity with SEQ ID No. 1 or SEQ        ID No. 2 or SEQ ID No. 3; or    -   b. a polynucleotide sequence shown herein as SEQ ID No. 4, SEQ        ID No. 5 or SEQ ID No. 6; or a nucleotide sequence which has at        least 97.7% (e.g. at least 98%, 98.5% or 99%) identity with SEQ        ID No. 4, SEQ ID No. 5 or SEQ ID No. 6.

A further aspect of the present invention provides a host cellcomprising a vector of the present invention or a nucleic acidcomprising (or consisting of) a nucleotide sequence shown herein as SEQID No. 4, SEQ ID No. 5 or SEQ ID No. 6; or a nucleotide sequence whichhas at least 97.7% (e.g. at least 98%, 98.5% or 99%) identity with SEQID No. 4, SEQ ID No. 5 or SEQ ID No. 6.

The present invention further relates to a method for degradingarabinoxylan-containing material comprising admixing anarabinoxylan-containing material with a xylanase, which xylanase is aGH10, fungal xylanase and degrades insoluble arabinoxylan (AXinsol) aswell as degrading the polymers, oligomers or combinations thereofproduced from the degradation of the AXinsol, and wherein the xylanasedegrades the polymers, oligomers or combinations thereof produced fromthe degradation of the AXinsol immediately or substantially immediatelyupon their production.

The present invention further relates to a method of degrading insolublearabinoxylan-containing material comprising admixing the material with axylanase comprising (or consisting of) a polypeptide sequence shownherein as SEQ ID No. 1, SEQ ID No. 2, SEQ ID No. 3, SEQ ID No. 9, SEQ IDNo. 10, SEQ ID No. 11 or SEQ ID No. 15, or a variant, homologue,fragment or derivative thereof having at least 75% identity (such as atleast 80%, 85%, 90%, 95%, 98% or 99% identity) with SEQ ID No. 1 or SEQID No. 2, SEQ ID No. 3, SEQ ID No. 9, SEQ ID No. 10, SEQ ID No. 11 orSEQ ID No. 15; or a polypeptide sequence which comprises SEQ ID No. 1,SEQ ID No. 2, SEQ ID No. 3, SEQ ID No. 9, SEQ ID No. 10, SEQ ID No. 11or SEQ ID No. 15 with a conservative substitution of at least one of theamino acids; or a xylanase which is encoded by a nucleotide sequenceshown herein as SEQ ID No. 4, SEQ ID No. 5, SEQ ID No. 6, SEQ ID No. 12,SEQ ID No. 13, SEQ ID No. 14, SEQ ID No. 16, SEQ ID No. 17 or SEQ ID No.18, or a nucleotide sequence which can hybridize to SEQ ID No. 4, SEQ IDNo. 5, SEQ ID No. 6, SEQ ID No. 12, SEQ ID No. 13, SEQ ID No. 14, SEQ IDNo. 16, SEQ ID No. 17 or SEQ ID No. 18 under high stringency conditions,or a nucleotide sequence which has at least 75% identity (such as atleast 80%, 85%, 90%, 95% or 98% identity) with SEQ ID No. 4, SEQ ID No.5, SEQ ID No. 6, SEQ ID No. 12, SEQ ID No. 13, SEQ ID No. 14, SEQ ID No.16, SEQ ID No. 17 or SEQ ID No. 18; or a nucleotide sequence whichdiffers from SEQ ID No. 4 or SEQ ID No. 5 or SEQ ID No. 6 or SEQ ID No.12 or SEQ ID No. 13 or SEQ ID No. 14 or SEQ ID No. 16 or SEQ ID No. 17or SEQ ID No. 18 due to the degeneracy of the genetic code.

The present invention further relates to a method for producing glucoseor starch (e.g. starch from wheat or glucose from starch), comprisingadmixing a xylan-containing material (preferably anarabinoxylan-containing material, such as an insolublearabinoxylan-containing material) with a xylanase comprising (orconsisting of) a polypeptide sequence shown herein as SEQ ID No. 1, SEQID No. 2, SEQ ID No. 3, SEQ ID No. 9, SEQ ID No. 10, SEQ ID No. 11, orSEQ ID No. 15, or a variant, homologue, fragment or derivative thereofhaving at least 75% Identity (such as at least 80%, 85%, 90%, 95%, 98%or 99% identity) with SEQ ID No. 1 or SEQ ID No. 2, SEQ ID No. 3, SEQ IDNo. 9, SEQ ID No. 10, SEQ ID No. 11 or SEQ ID No. 15; or a polypeptidesequence which comprises SEQ ID No. 1, SEQ ID No. 2, SEQ ID No. 3, SEQID No. 9, SEQ ID No. 10, SEQ ID No. 11, or SEQ ID No. 15 with aconservative substitution of at least one of the amino acids; or axylanase which is encoded by a nucleotide sequence shown herein as SEQID No. 4, SEQ ID No. 5, SEQ ID No. 6, SEQ ID No. 12, SEQ ID No. 13, SEQID No. 14, SEQ ID No. 16, SEQ ID No. 17 or SEQ ID No. 18, or anucleotide sequence which can hybridize to SEQ ID No. 4, SEQ ID No. 5,SEQ ID No. 6, SEQ ID No. 12, SEQ ID No. 13, SEQ ID No. 14, SEQ ID No.16, SEQ ID No. 17 or SEQ ID No. 18 under high stringency conditions, ora nucleotide sequence which has at least 75% identity (such as at least80%, 85%, 90%, 95% or 98% identity) with SEQ ID No. 4, SEQ ID No. 5, SEQID No. 6, SEQ ID No. 12, SEQ ID No. 13, SEQ ID No. 14, SEQ ID No. 16,SEQ ID No. 17 or SEQ ID No. 18, or a nucleotide sequence which differsfrom SEQ ID No. 4 or SEQ ID No. 5 or SEQ ID No. 6 or SEQ ID No. 12 orSEQ ID No. 13 or SEQ ID No. 14 or SEQ ID No. 16 or SEQ ID No. 17 or SEQID No. 18 due to the degeneracy of the genetic code.

In one embodiment the xylan-containing material further comprisesstarch.

Use of a xylanase comprising (or consisting of) a polypeptide sequenceshown herein as SEQ ID No. 1, SEQ ID No. 2, SEQ ID No. 3, SEQ ID No. 9,SEQ ID No. 10, SEQ ID No. 11 or SEQ ID No. 15, or a variant, homologue,fragment or derivative thereof having at least 75% identity (such as atleast 80%, 85%, 90%, 95%, 98% or 99% identity) with SEQ ID No. 1, SEQ IDNo. 2, SEQ ID No. 3, SEQ ID No. 9, SEQ ID No. 10, SEQ ID No. 11 or SEQID No. 15; or a polypeptide sequence which comprises SEQ ID No. 1, SEQID No. 2, SEQ ID No. 3, SEQ ID No. 9, SEQ ID No. 10, SEQ ID No. 11 orSEQ ID No. 15 with a conservative substitution of at least one of theamino acids; or a xylanase which is encoded by a nucleotide sequenceshown herein as SEQ ID No. 4, SEQ ID No. 5, SEQ ID No. 6, SEQ ID No. 12,SEQ ID No. 13, SEQ ID No. 14, SEQ ID No. 16, SEQ ID No. 17 or SEQ ID No.18, or a nucleotide sequence which can hybridize to SEQ ID No. 4, SEQ IDNo. 5, SEQ ID No. 6, SEQ ID No. 12, SEQ ID No. 13, SEQ ID No. 14, SEQ IDNo. 16, SEQ ID No. 17 or SEQ ID No. 18 under high stringency conditions,or a nucleotide sequence which has at least 75% identity (such as atleast 80%, 85%, 90%, 95% or 98% identity) with SEQ ID No. 4, SEQ ID No.5, SEQ ID No. 6, SEQ ID No. 12, SEQ ID No. 13, SEQ ID No. 14, SEQ ID No.16, SEQ ID No. 17 or SEQ ID No. 18, or a nucleotide sequence whichdiffers from SEQ ID No. 4 or SEQ ID No. 5 or SEQ ID No. 6 or SEQ ID No.12 or SEQ ID No. 13 or SEQ ID No. 14 or SEQ ID No. 16 or SEQ ID No. 17or SEQ ID No. 18 due to the degeneracy of the genetic code for degradinga xylan-containing material (preferably an arabinoxylan-containingmaterial, preferably an insoluble arabinoxylan-containing material),optionally without increasing the viscosity or also decreasing theviscosity of the reaction mixture.

According to another aspect of the present invention, there is provideda method for improving the performance of a subject or for improvingdigestibility of a raw material in a feed (e.g. nutrient digestibility)or for improving feed efficiency in a subject or for reducing theviscosity of the intestinal content, the method comprising administeringa subject with a xylanase comprising (or consisting of) a polypeptidesequence shown herein as SEQ ID No. 1, SEQ ID No. 2, SEQ ID No. 3, SEQID No. 9, SEQ ID No. 10, SEQ ID No. 11 or SEQ ID No. 15, or a variant,homologue, fragment or derivative thereof having at least 75% identity(such as at least 80%, 85%, 90%, 95%, 98% or 99% identity) with SEQ IDNo. 1, SEQ ID No. 2, SEQ ID No. 3, SEQ ID No. 9, SEQ ID No. 10, SEQ IDNo. 11 or SEQ ID No. 15; or a polypeptide sequence which comprises SEQID No. 1, SEQ ID No. 2, SEQ ID No. 3, SEQ ID No. 9, SEQ ID No. 10, SEQID No. 11 or SEQ ID No. 15 with a conservative substitution of at leastone of the amino acids; or a xylanase which is encoded by a nucleotidesequence shown herein as SEQ ID No. 4, SEQ ID No. 5, SEQ ID No. 6, SEQID No. 12, SEQ ID No. 13, SEQ ID No. 14, SEQ ID No. 16, SEQ ID No. 17 orSEQ ID No. 18, or a nucleotide sequence which can hybridize to SEQ IDNo. 4, SEQ ID No. 5, SEQ ID No. 6, SEQ ID No. 12, SEQ ID No. 13, SEQ IDNo. 14, SEQ ID No. 16, SEQ ID No. 17 or SEQ ID No. 18 under highstringency conditions, or a nucleotide sequence which has at least 75%identity (such as at least 80%, 85%, 90%, 95% or 98% identity) with SEQID No. 4, SEQ ID No. 5, SEQ ID No. 6, SEQ ID No. 12, SEQ ID No. 13, SEQID No. 14, SEQ ID No. 16, SEQ ID No. 17 or SEQ ID No. 18, or anucleotide sequence which differs from SEQ ID No. 4 or SEQ ID No. 5 orSEQ ID No. 6 or SEQ ID No. 12 or SEQ ID No. 13 or SEQ ID No. 14 or SEQID No. 16 or SEQ ID No. 17 or SEQ ID No. 18 due to the degeneracy of thegenetic code or administering a subject with a xylanase obtainable (orobtained) from Fusarium verticilloides.

A yet further aspect of the present invention is use of a xylanasecomprising (or consisting of) a polypeptide sequence shown herein as SEQID No. 1, SEQ ID No. 2, SEQ ID No. 3, SEQ ID No. 9, SEQ ID No. 10, SEQID No. 11 or SEQ ID No. 15, or a variant, homologue, fragment orderivative thereof having at least 75% identity (such as at least 80%,85%, 90%, 95%, 98% or 99% identity) with SEQ ID No. 1 or SEQ ID No. 2 orSEQ ID No. 3 or SEQ ID No. 9 or SEQ ID No. 10, SEQ ID No. 11 or SEQ IDNo. 15; or a polypeptide sequence which comprises SEQ ID No. 1, SEQ IDNo. 2, SEQ ID No. 3, SEQ ID No. 9, SEQ ID No. 10, SEQ ID No. 11 or SEQID No. 15 with a conservative substitution of at least one of the aminoacids; or a xylanase which is encoded by a nucleotide sequence shownherein as SEQ ID No. 4, SEQ ID No. 5, SEQ ID No. 6, SEQ ID No. 12, SEQID No. 13, SEQ ID No. 14, SEQ ID No. 16, SEQ ID No. 17 or SEQ ID No. 18,or a nucleotide sequence which can hybridize to SEQ ID No. 4, SEQ ID No.5, SEQ ID No. 6, SEQ ID No. 12, SEQ ID No. 13, SEQ ID No. 14, SEQ ID No.16, SEQ ID No. 17 or SEQ ID No. 18 under high stringency conditions, ora nucleotide sequence which has at least 75% identity (such as at least80%, 85%, 90%, 95% or 98% identity) with SEQ ID No. 4, SEQ ID No. 5, SEQID No. 6, SEQ ID No. 12, SEQ ID No. 13, SEQ ID No. 14, SEQ ID No. 16,SEQ ID No. 17 or SEQ ID No. 18, or a nucleotide sequence which differsfrom SEQ ID No. 4 or SEQ ID No. 5 or SEQ ID No. 6 or SEQ ID No. 12 orSEQ ID No. 13 or SEQ ID No. 14 or SEQ ID No. 16 or SEQ ID No. 17 or SEQID No. 18 due to the degeneracy of the genetic code or use of a xylanaseobtainable (or obtained) from Fusarium verticilloides for improving theperformance of a subject or for improving digestibility of a rawmaterial in a feed (e.g. nutrient digestibility) or for improving feedefficiency in a subject or for reducing the viscosity of the intestinalcontent.

In another aspect the present invention provides a feed additivecomposition comprising (or consisting essentially of or consisting of) axylanase comprising (or consisting of) a polypeptide sequence shownherein as SEQ ID No. 1, SEQ ID No. 2, SEQ ID No. 3, SEQ ID No. 9, SEQ IDNo. 10, SEQ ID No. 11, or SEQ ID No. 15 or a variant, homologue,fragment or derivative thereof having at least 75% identity (such as atleast 80%, 85%, 90%, 95%, 98% or 99% identity) with SEQ ID No. 1 or SEQID No. 2 or SEQ ID No. 3 or SEQ ID No. 9 or SEQ ID No. 10, SEQ ID No. 11or SEQ ID No. 15; or a polypeptide sequence which comprises SEQ ID No.1, SEQ ID No. 2, SEQ ID No. 3, SEQ ID No. 9, SEQ ID No. 10, SEQ ID No.11 or SEQ ID No. 15 with a conservative substitution of at least one ofthe amino acids; or a xylanase which is encoded by a nucleotide sequenceshown herein as SEQ ID No. 4, SEQ ID No. 5, SEQ ID No. 6, SEQ ID No. 12,SEQ ID No. 13, SEQ ID No. 14, SEQ ID No. 16, SEQ ID No. 17 or SEQ ID No.18, or a nucleotide sequence which can hybridize to SEQ ID No. 4, SEQ IDNo. 5, SEQ ID No. 6, SEQ ID No. 12, SEQ ID No. 13, SEQ ID No. 14, SEQ IDNo. 16, SEQ ID No. 17 or SEQ ID No. 18 under high stringency conditions,or a nucleotide sequence which has at least 75% identity (such as atleast 80%, 85%, 90%, 95% or 98% identity) with SEQ ID No. 4, SEQ ID No.5, SEQ ID No. 6, SEQ ID No. 12, SEQ ID No. 13, SEQ ID No. 14, SEQ ID No.16, SEQ ID No. 17 or SEQ ID No. 18, or a nucleotide sequence whichdiffers from SEQ ID No. 4 or SEQ ID No. 5 or SEQ ID No. 6 or SEQ ID No.12 or SEQ ID No. 13 or SEQ ID No. 14 or SEQ ID No. 16 or SEQ ID No. 17or SEQ ID No. 18 due to the degeneracy of the genetic code; or axylanase obtainable (or obtained) from Fusarium verticilloides.

In a further aspect the present invention provides a kit comprisingxylanase comprising (or consisting of) a polypeptide sequence shownherein as SEQ ID No. 1, SEQ ID No. 2, SEQ ID No. 3, SEQ ID No. 9, SEQ IDNo. 10, SEQ ID No. 11 or SEQ ID No. 15, or a variant, homologue,fragment or derivative thereof having at least 75% identity (such as atleast 80%, 85%, 90%, 95%, 98% or 99% identity) with SEQ ID No. 1 or SEQID No. 2 or SEQ ID No. 3 or SEQ ID No. 9 or SEQ ID No. 10 or SEQ ID No.11 or SEQ ID No. 15; or a polypeptide sequence which comprises SEQ IDNo. 1, SEQ ID No. 2, SEQ ID No. 3, SEQ ID No. 9, SEQ ID No. 10, SEQ IDNo. 11 or SEQ ID No. 15 with a conservative substitution of at least oneof the amino acids; or a xylanase which is encoded by a nucleotidesequence shown herein as SEQ ID No. 4, SEQ ID No. 5, SEQ ID No. 6, SEQID No. 12, SEQ ID No. 13, SEQ ID No. 14, SEQ ID No. 16, SEQ ID No. 17 orSEQ ID No. 18, or a nucleotide sequence which can hybridize to SEQ IDNo. 4, SEQ ID No. 5, SEQ ID No. 6, SEQ ID No. 12, SEQ ID No. 13, SEQ IDNo. 14, SEQ ID No. 16, SEQ ID No. 17 or SEQ ID No. 18 under highstringency conditions, or a nucleotide sequence which has at least 75%identity (such as at least 80%, 85%, 90%, 95% or 98% identity) with SEQID No. 4, SEQ ID No. 5, SEQ ID No. 6, SEQ ID No. 12, SEQ ID No. 13, SEQID No. 14, SEQ ID No. 16, SEQ ID No. 17 or SEQ ID No. 18, or anucleotide sequence which differs from SEQ ID No. 4 or SEQ ID No. 5 orSEQ ID No. 6 or SEQ ID No. 12 or SEQ ID No. 13 or SEQ ID No. 14 or SEQID No. 16 or SEQ ID No. 17 or SEQ ID No. 18 due to the degeneracy of thegenetic code and instructions for administration.

In a yet further aspect the present invention provides a feedstuffcomprising a feed additive composition comprising (or consistingessentially of or consisting of) a xylanase comprising (or consistingof) a polypeptide sequence shown herein as SEQ ID No. 1, SEQ ID No. 2,SEQ ID No. 3, SEQ ID No. 9, SEQ ID No. 10, SEQ ID No. 11 or SEQ ID No.15, or a variant, homologue, fragment or derivative thereof having atleast 75% identity (such as at least 80%, 85%, 90%, 95%, 98% or 99%identity) with SEQ ID No. 1 or SEQ ID No. 2 or SEQ ID No. 3 or SEQ IDNo. 9 or SEQ ID No. 10 or SEQ ID No. 11 or SEQ ID No. 15; or apolypeptide sequence which comprises SEQ ID No. 1, SEQ ID No. 2, SEQ IDNo. 3, SEQ ID No. 9, SEQ ID No. 10, SEQ ID No. 11 or SEQ ID No. 15 witha conservative substitution of at least one of the amino acids; or axylanase which is encoded by a nucleotide sequence shown herein as SEQID No. 4, SEQ ID No. 5, SEQ ID No. 6, SEQ ID No. 12, SEQ ID No. 13, SEQID No. 14, SEQ ID No. 16, SEQ ID No. 17 or SEQ ID No. 18, or anucleotide sequence which can hybridize to SEQ ID No. 4, SEQ ID No. 5,SEQ ID No. 6, SEQ ID No. 12, SEQ ID No. 13, SEQ ID No. 14, SEQ ID No.16, SEQ ID No. 17 or SEQ ID No. 18 under high stringency conditions, ora nucleotide sequence which has at least 75% identity (such as at least80%, 85%, 90%, 95% or 98% identity) with SEQ ID No. 4, SEQ ID No. 5, SEQID No. 6, SEQ ID No. 12, SEQ ID No. 13, SEQ ID No. 14, SEQ ID No. 16,SEQ ID No. 17 or SEQ ID No. 18, or a nucleotide sequence which differsfrom SEQ ID No. 4 or SEQ ID No. 5 or SEQ ID No. 6 or SEQ ID No. 12 orSEQ ID No. 13 or SEQ ID No. 14 or SEQ ID No. 16 or SEQ ID No. 17 or SEQID No. 18 due to the degeneracy of the genetic code, or a xylanaseobtainable (or obtained) from Fusarium verticilloides.

A premix comprising a feed additive composition comprising (orconsisting essentially of or consisting of) a xylanase comprising (orconsisting of) a polypeptide sequence shown herein as SEQ ID No. 1, SEQID No. 2, SEQ ID No. 3, SEQ ID No. 9, SEQ ID No. 10, SEQ ID No. 11 orSEQ ID No. 15, or a variant, homologue, fragment or derivative thereofhaving at least 75% identity (such as at least 80%, 85%, 90%, 95%, 98%or 99% identity) with SEQ ID No. 1 or SEQ ID No. 2 or SEQ ID No. 3 orSEQ ID No. 9 or SEQ ID No. 10 or SEQ ID No. 11 or SEQ ID No. 15; or apolypeptide sequence which comprises SEQ ID No. 1, SEQ ID No. 2, SEQ IDNo. 3, SEQ ID No. 9, SEQ ID No. 10, SEQ ID No. 11 or SEQ ID No. 15 witha conservative substitution of at least one of the amino acids; or axylanase which is encoded by a nucleotide sequence shown herein as SEQID No. 4, SEQ ID No. 5, SEQ ID No. 6, SEQ ID No. 12, SEQ ID No. 13, SEQID No. 14, SEQ ID No. 16, SEQ ID No. 17 or SEQ ID No. 18, or anucleotide sequence which can hybridize to SEQ ID No. 4, SEQ ID No. 5,SEQ ID No. 6, SEQ ID No. 12, SEQ ID No. 13, SEQ ID No. 14, SEQ ID No.16, SEQ ID No. 17 or SEQ ID No. 18 under high stringency conditions, ora nucleotide sequence which has at least 75% identity (such as at least80%, 85%, 90%, 95% or 98% Identity) with SEQ ID No. 4, SEQ ID No. 5, SEQID No. 6, SEQ ID No. 12, SEQ ID No. 13, SEQ ID No. 14, SEQ ID No. 16,SEQ ID No. 17 or SEQ ID No. 18, or a nucleotide sequence which differsfrom SEQ ID No. 4 or SEQ ID No. 5 or SEQ ID No. 6 or SEQ ID No. 12 orSEQ ID No. 13 or SEQ ID No. 14 or SEQ ID No. 16 or SEQ ID No. 17 or SEQID No. 18 due to the degeneracy of the genetic code; or a xylanaseobtainable (or obtained) from Fusarium verticilloides, and at least onemineral and/or at least one vitamin.

In another aspect, the present invention provides a method of preparinga feedstuff comprising admixing a feed component with a feed additivecomposition comprising (or consisting essentially of or consisting of) axylanase comprising (or consisting of) a polypeptide sequence shownherein as SEQ ID No. 1, SEQ ID No. 2, SEQ ID No. 3, SEQ ID No. 9, SEQ IDNo. 10, SEQ ID No. 11 or SEQ ID No. 15, or a variant, homologue,fragment or derivative thereof having at least 75% identity (such as atleast 80%, 85%, 90%, 95%, 98% or 99% identity) with SEQ ID No. 1 or SEQID No. 2 or SEQ ID No. 3 or SEQ ID No. 9 or SEQ ID No. 10 or SEQ ID No.11 or SEQ ID No. 15; or a polypeptide sequence which comprises SEQ IDNo. 1, SEQ ID No. 2, SEQ ID No. 3, SEQ ID No. 9, SEQ ID No. 10, SEQ IDNo. 11 or SEQ ID No. 15 with a conservative substitution of at least oneof the amino acids; or a xylanase which is encoded by a nucleotidesequence shown herein as SEQ ID No. 4, SEQ ID No. 5, SEQ ID No. 6, SEQID No. 12, SEQ ID No. 13, SEQ ID No. 14, SEQ ID No. 16, SEQ ID No. 17 orSEQ ID No. 18, or a nucleotide sequence which can hybridize to SEQ IDNo. 4, SEQ ID No. 5, SEQ ID No. 6, SEQ ID No. 12, SEQ ID No. 13, SEQ IDNo. 14, SEQ ID No. 16, SEQ ID No. 17 or SEQ ID No. 18 under highstringency conditions, or a nucleotide sequence which has at least 75%identity (such as at least 80%, 85%, 90%, 95% or 98% identity) with SEQID No. 4, SEQ ID No. 5, SEQ ID No. 6, SEQ ID No. 12, SEQ ID No. 13, SEQID No. 14, SEQ ID No. 16, SEQ ID No. 17 or SEQ ID No. 18, or anucleotide sequence which differs from SEQ ID No. 4 or SEQ ID No. 5 orSEQ ID No. 6 or SEQ ID No. 12 or SEQ ID No. 13 or SEQ ID No. 14 or SEQID No. 16 or SEQ ID No. 17 or SEQ ID No. 18 due to the degeneracy of thegenetic code, or a xylanase obtainable (or obtained) from Fusariumverticilloides.

According to another aspect of this present invention, there is provideda method for degrading grain-based material (including whole grains orpartial grains or malted grains, e.g. malted barley) and reducingviscosity in the reaction medium (e.g. grain mash), the methodcomprising admixing said grain-based material with a xylanase comprising(or consisting of) a polypeptide sequence shown herein as SEQ ID No. 1,SEQ ID No. 2, SEQ ID No. 3, SEQ ID No. 9, SEQ ID No. 10, SEQ ID No. 11or SEQ ID No. 15, or a variant, homologue, fragment or derivativethereof having at least 75% identity (such as at least 80%, 85%, 90%,95%, 98% or 99% identity) with SEQ ID No. 1 or SEQ ID No. 2 or SEQ IDNo. 3 or SEQ ID No. 9 or SEQ ID No. 10 or SEQ ID No. 11 or SEQ ID No.15; or a polypeptide sequence which comprises SEQ ID No. 1, SEQ ID No.2, SEQ ID No. 3, SEQ ID No. 9, SEQ ID No. 10, SEQ ID No. 11 or SEQ IDNo. 15 with a conservative substitution of at least one of the aminoacids; or a xylanase which is encoded by a nucleotide sequence shownherein as SEQ ID No. 4, SEQ ID No. 5, SEQ ID No. 6, SEQ ID No. 12, SEQID No. 13, SEQ ID No. 14, SEQ ID No. 16, SEQ ID No. 17 or SEQ ID No. 18,or a nucleotide sequence which can hybridize to SEQ ID No. 4, SEQ ID No.5, SEQ ID No. 6, SEQ ID No. 12, SEQ ID No. 13, SEQ ID No. 14, SEQ ID No.16, SEQ ID No. 17 or SEQ ID No. 18 under high stringency conditions, ora nucleotide sequence which has at least 75% identity (such as at least80%, 85%, 90%, 95% or 98% identity) with SEQ ID No. 4, SEQ ID No. 5, SEQID No. 6, SEQ ID No. 12, SEQ ID No. 13, SEQ ID No. 14, SEQ ID No. 16,SEQ ID No. 17 or SEQ ID No. 18, or a nucleotide sequence which differsfrom SEQ ID No. 4 or SEQ ID No. 5 or SEQ ID No. 6 or SEQ ID No. 12 orSEQ ID No. 13 or SEQ ID No. 14 or SEQ ID No. 16 or SEQ ID No. 17 or SEQID No. 18 due to the degeneracy of the genetic code, or a xylanaseobtainable (or obtained) from Fusarium verticilloides.

A yet further aspect of the present invention is use of a xylanasecomprising (or consisting of) a polypeptide sequence shown herein as SEQID No. 1, SEQ ID No. 2, SEQ ID No. 3, SEQ ID No. 9, SEQ ID No. 10, SEQID No. 11 or SEQ ID No. 15, or a variant, homologue, fragment orderivative thereof having at least 75% identity (such as at least 80%,85%, 90%, 95%, 98% or 99% identity) with SEQ ID No. 1 or SEQ ID No. 2 orSEQ ID No. 3 or SEQ ID No. 9 or SEQ ID No. 10 or SEQ ID No. 11 or SEQ IDNo. 15; or a xylanase which is encoded by a nucleotide sequence shownherein as SEQ ID No. 4, SEQ ID No. 5, SEQ ID No. 6, SEQ ID No. 12, SEQID No. 13, SEQ ID No. 14, SEQ ID No. 16, SEQ ID No. 17 or SEQ ID No. 18,or a nucleotide sequence which can hybridize to SEQ ID No. 4, SEQ ID No.5, SEQ ID No. 6, SEQ ID No. 12, SEQ ID No. 13, SEQ ID No. 14, SEQ ID No.16, SEQ ID No. 17 or SEQ ID No. 18 under high stringency conditions, ora nucleotide sequence which has at least 75% identity (such as at least80%, 85%, 90%, 95% or 98% identity) with SEQ ID No. 4, SEQ ID No. 5, SEQID No. 6, SEQ ID No. 12, SEQ ID No. 13, SEQ ID No. 14, SEQ ID No. 16,SEQ ID No. 17 or SEQ ID No. 18, or a nucleotide sequence which differsfrom SEQ ID No. 4 or SEQ ID No. 5 or SEQ ID No. 6 or SEQ ID No. 12 orSEQ ID No. 13 or SEQ ID No. 14 or SEQ ID No. 16 or SEQ ID No. 17 or SEQID No. 18 due to the degeneracy of the genetic code, or use of axylanase obtainable (or obtained) from Fusarium verticilloides fordegrading grain-based material (including whole grains or partial grainsor malted grains, e.g. malted barley) whilst reducing viscosity in thereaction medium (e.g. grain mash).

According to another aspect of the present invention, there is provideda method for producing biofuel (e.g. bioethanol) or a biochemical (e.g.bio-based isoprene) comprising admixing a grain-based material with axylanase comprising (or consisting of) a polypeptide sequence shownherein as SEQ ID No. 1, SEQ ID No. 2, SEQ ID No. 3, SEQ ID No. 9, SEQ IDNo. 10, SEQ ID No. 11 or SEQ ID No. 15, or a variant, homologue,fragment or derivative thereof having at least 75% identity (such as atleast 80%, 85%, 90%, 95%, 98% or 99% identity) with SEQ ID No. 1 or SEQID No. 2 or SEQ ID No. 3 or SEQ ID No. 9 or SEQ ID No. 10 or SEQ ID No.11 or SEQ ID No. 15; or a polypeptide sequence which comprises SEQ IDNo. 1, SEQ ID No. 2, SEQ ID No. 3, SEQ ID No. 9, SEQ ID No. 10, SEQ IDNo. 11 or SEQ ID No. 15 with a conservative substitution of at least oneof the amino acids; or a xylanase which is encoded by a nucleotidesequence shown herein as SEQ ID No. 4, SEQ ID No. 5, SEQ ID No. 6, SEQID No. 12, SEQ ID No. 13, SEQ ID No. 14, SEQ ID No. 16, SEQ ID No. 17 orSEQ ID No. 18, or a nucleotide sequence which can hybridize to SEQ IDNo. 4, SEQ ID No. 5, SEQ ID No. 6, SEQ ID No. 12, SEQ ID No. 13, SEQ IDNo. 14, SEQ ID No. 16, SEQ ID No. 17 or SEQ ID No. 18 under highstringency conditions, or a nucleotide sequence which has at least 75%identity (such as at least 80%, 85%, 90%, 95% or 98% identity) with SEQID No. 4, SEQ ID No. 5, SEQ ID No. 6, SEQ ID No. 12, SEQ ID No. 13, SEQID No. 14, SEQ ID No. 16, SEQ ID No. 17 or SEQ ID No. 18, or anucleotide sequence which differs from SEQ ID No. 4 or SEQ ID No. 5 orSEQ ID No. 6 or SEQ ID No. 12 or SEQ ID No. 13 or SEQ ID No. 14 or SEQID No. 16 or SEQ ID No. 17 or SEQ ID No. 18 due to the degeneracy of thegenetic code, or a xylanase obtainable (or obtained) from Fusariumverticilloides.

A yet further aspect of the present invention is use of a xylanasecomprising (or consisting of) a polypeptide sequence shown herein as SEQID No. 1, SEQ ID No. 2, SEQ ID No. 3, SEQ ID No. 9, SEQ ID No. 10, SEQID No. 11 or SEQ ID No. 15, or a variant, homologue, fragment orderivative thereof having at least 75% identity (such as at least 80%,85%, 90%, 95%, 98% or 99% identity) with SEQ ID No. 1 or SEQ ID No. 2 orSEQ ID No. 3 or SEQ ID No. 9 or SEQ ID No. 10 or SEQ ID No. 11 or SEQ IDNo. 15; or a xylanase which is encoded by a nucieotide.sequence shownherein as SEQ ID No. 4, SEQ ID No. 5, SEQ ID No. 6, SEQ ID No. 12, SEQID No. 13, SEQ ID No. 14, SEQ ID No. 16, SEQ ID No. 17 or SEQ ID No. 18,or a nucleotide sequence which can hybridize to SEQ ID No. 4, SEQ ID No.5, SEQ ID No. 6, SEQ ID No. 12, SEQ ID No. 13, SEQ ID No. 14, SEQ ID No.16, SEQ ID No. 17 or SEQ ID No. 18 under high stringency conditions, ora nucleotide sequence which has at least 75% identity (such as at least80%, 85%, 90%, 95% or 98% identity) with SEQ ID No. 4, SEQ ID No. 5, SEQID No. 6, SEQ ID No. 12, SEQ ID No. 13, SEQ ID No. 14, SEQ ID No. 16,SEQ ID No. 17 or SEQ ID No. 18, or a nucleotide sequence which differsfrom SEQ ID No. 4 or SEQ ID No. 5 or SEQ ID No. 6 or SEQ ID No. 12 orSEQ ID No. 13 or SEQ ID No. 14 or SEQ ID No. 16 or SEQ ID No. 17 or SEQID No. 18 due to the degeneracy of the genetic code, or use of axylanase obtainable (or obtained) from Fusarium verticilloides forproducing biofuel (e.g. bioethanol) or a biochemical (e.g. bio-basedisoprene).

According to another aspect of the present invention, there is provideda method for separating a cereal flour (e.g. wheat flour) into starchand gluten fractions the method comprising admixing a cereal flour (e.g.wheat flour), water and a xylanase comprising (or consisting of) apolypeptide sequence shown herein as SEQ ID No. 1, SEQ ID No. 2, SEQ IDNo. 3, SEQ ID No. 9, SEQ ID No. 10, SEQ ID No. 11 or SEQ ID No. 15, or avariant, homologue, fragment or derivative thereof having at least 75%identity (such as at least 80%, 85%, 90%, 95%, 98% or 99% identity) withSEQ ID No. 1 or SEQ ID No. 2 or SEQ ID No. 3 or SEQ ID No. 9 or SEQ IDNo. 10 or SEQ ID No. 11 or SEQ ID No. 15; or a polypeptide sequencewhich comprises SEQ ID No. 1, SEQ ID No. 2, SEQ ID No. 3, SEQ ID No. 9,SEQ ID No. 10, SEQ ID No. 11 or SEQ ID No. 15 with a conservativesubstitution of at least one of the amino acids; or a xylanase which isencoded by a nucleotide sequence shown herein as SEQ ID No. 4, SEQ IDNo. 5, SEQ ID No. 6, SEQ ID No. 12, SEQ ID No. 13, SEQ ID No. 14, SEQ IDNo. 16, SEQ ID No. 17 or SEQ ID No. 18, or a nucleotide sequence whichcan hybridize to SEQ ID No. 4, SEQ ID No. 5, SEQ ID No. 6, SEQ ID No.12, SEQ ID No. 13, SEQ ID No. 14, SEQ ID No. 16, SEQ ID No. 17 or SEQ IDNo. 18 under high stringency conditions, or a nucleotide sequence whichhas at least 75% identity (such as at least 80%, 85%, 90%, 95% or 98%identity) with SEQ ID No. 4, SEQ ID No. 5, SEQ ID No. 6, SEQ ID No. 12,SEQ ID No. 13, SEQ ID No. 14, SEQ ID No. 16, SEQ ID No. 17 or SEQ ID No.18, or a nucleotide sequence which differs from SEQ ID No. 4 or SEQ IDNo. 5 or SEQ ID No. 6 or SEQ ID No. 12 or SEQ ID No. 13 or SEQ ID No. 14or SEQ ID No. 16 or SEQ ID No. 17 or SEQ ID No. 18 due to the degeneracyof the genetic code, or a xylanase obtainable (or obtained) fromFusarium verticilloides.

A yet further aspect of the present invention provides the use of axylanase comprising (or consisting of) a polypeptide sequence shownherein as SEQ ID No. 1, SEQ ID No. 2, SEQ ID No. 3, SEQ ID No. 9, SEQ IDNo. 10, SEQ ID No. 11 or SEQ ID No. 15, or a variant, homologue,fragment or derivative thereof having at least 75% identity (such as atleast 80%, 85%, 90%, 95%, 98% or 99% Identity) with SEQ ID No. 1 or SEQID No. 2 or SEQ ID No. 3 or SEQ ID No. 9 or SEQ ID No. 10 or SEQ ID No.11 or SEQ ID No. 15; or a polypeptide sequence which comprises SEQ IDNo. 1, SEQ ID No. 2, SEQ ID No. 3, SEQ ID No. 9, SEQ ID No. 10, SEQ IDNo. 11 or SEQ ID No. 15 with a conservative substitution of at least oneof the amino acids; or a xylanase which is encoded by a nucleotidesequence shown herein as SEQ ID No. 4, SEQ ID No. 5, SEQ ID No. 6, SEQID No. 12, SEQ ID No. 13, SEQ ID No. 14, SEQ ID No. 16, SEQ ID No. 17 orSEQ ID No. 18, or a nucleotide sequence which can hybridize to SEQ IDNo. 4, SEQ ID No. 5, SEQ ID No. 6, SEQ ID No. 12, SEQ ID No. 13, SEQ IDNo. 14, SEQ ID No. 16, SEQ ID No. 17 or SEQ ID No. 18 under highstringency conditions, or a nucleotide sequence which has at least 75%identity (such as at least 80%, 85%, 90%, 95% or 98% identity) with SEQID No. 4, SEQ ID No. 5, SEQ ID No. 6, SEQ ID No. 12, SEQ ID No. 13, SEQID No. 14, SEQ ID No. 16, SEQ ID No. 17 or SEQ ID No. 18, or anucleotide sequence which differs from,SEQ ID No. 4 or SEQ ID No. 5 orSEQ ID No. 6 or SEQ ID No. 12 or SEQ ID No. 13 or SEQ ID No. 14 or SEQID No. 16 or SEQ ID No. 17 or SEQ ID No. 18 due to the degeneracy of thegenetic code, or use of a xylanase obtainable (or obtained) fromFusarium verticilloides for separating a cereal flour (e.g. wheat flour)into starch and gluten fractions whilst reducing viscosity in thereaction medium (e.g. grain mash).

In a further aspect there is provided the use of the xylanase comprising(or consisting of) a polypeptide sequence shown herein as SEQ ID No. 1,SEQ ID No. 2, SEQ ID No. 3, SEQ ID No. 9, SEQ ID No. 10, SEQ ID No. 11or SEQ ID No. 15, or a variant, homologue, fragment or derivativethereof having at least 75% identity (such as at least 80%, 85%, 90%,95%, 98% or 99% identity) with SEQ ID No. 1 or SEQ ID No. 2, SEQ ID No.3, SEQ ID No. 9, SEQ ID No. 10, SEQ ID No. 11 or SEQ ID No. 15; or apolypeptide sequence which comprises SEQ ID No. 1, SEQ ID No. 2, SEQ IDNo. 3, SEQ ID No. 9, SEQ ID No. 10, SEQ ID No. 11 or SEQ ID No. 15 witha conservative substitution of at least one of the amino acids; or axylanase which is encoded by a nucleotide sequence shown herein as SEQID No. 4, SEQ ID No. 5, SEQ ID No. 6, SEQ ID No. 12, SEQ ID No. 13, SEQID No. 14, SEQ ID No. 16, SEQ ID No. 17 or SEQ ID No. 18, or anucleotide sequence which can hybridize to SEQ ID No. 4, SEQ ID No. 5,SEQ ID No. 6, SEQ ID No. 12, SEQ ID No. 13, SEQ ID No. 14, SEQ ID No.16, SEQ ID No. 17 or SEQ ID No. 18 under high stringency conditions, ora nucleotide sequence which has at least 75% identity (such as at least80%, 85%, 90%, 95% or 98% identity) with SEQ ID No. 4, SEQ ID No. 5, SEQID No. 6, SEQ ID No. 12, SEQ ID No. 13, SEQ ID No. 14, SEQ ID No. 16,SEQ ID No. 17 or SEQ ID No. 18; or a nucleotide sequence which differsfrom SEQ ID No. 4 or SEQ ID No. 5 or SEQ ID No. 6 or SEQ ID No. 12 orSEQ ID No. 13 or SEQ ID No. 14 or SEQ ID No. 16 or SEQ ID No. 17 or SEQID No. 18 due to the degeneracy of the genetic code, in the productionof a fermented beverage, such as a beer.

In a yet further aspect, the present invention provides a method ofproducing a fermented beverage (e.g. beer) comprising the step ofcontacting a mash and/or a wort with a xylanase comprising (orconsisting of) a polypeptide sequence shown herein as SEQ ID No. 1, SEQID No. 2, SEQ ID No. 3, SEQ ID No. 9, SEQ ID No. 10, SEQ ID No. 11 orSEQ ID No. 15, or a variant, homologue, fragment or derivative thereofhaving at least 75% identity (such as at least 80%, 85%, 90%, 95%, 98%or 99% identity) with SEQ ID No. 1 or SEQ ID No. 2, SEQ ID No. 3, SEQ IDNo. 9, SEQ ID No. 10, SEQ ID No. 11 or SEQ ID No. 15; or a polypeptidesequence which comprises SEQ ID No. 1, SEQ ID No. 2, SEQ ID No. 3, SEQID No. 9, SEQ ID No. 10, SEQ ID No. 11 or SEQ ID No. 15 with aconservative substitution of at least one of the amino acids; or axylanase which is encoded by a nucleotide sequence shown herein as SEQID No. 4, SEQ ID No. 5, SEQ ID No. 6, SEQ ID No. 12, SEQ ID No. 13, SEQID No. 14, SEQ ID No. 16, SEQ ID No. 17 or SEQ ID No. 18, or anucleotide sequence which can hybridize to SEQ ID No. 4, SEQ ID No. 5,SEQ ID No. 6, SEQ ID No. 12, SEQ ID No. 13, SEQ ID No. 14, SEQ ID No.16, SEQ ID No. 17 or SEQ ID No. 18 under high stringency conditions, ora nucleotide sequence which has at least 75% identity (such as at least80%, 85%, 90%, 95% or 98% identity) with SEQ ID No. 4, SEQ ID No. 5, SEQID No. 6, SEQ ID No. 12, SEQ ID No. 13, SEQ ID No. 14, SEQ ID No. 16,SEQ ID No. 17 or SEQ ID No. 18; or a nucleotide sequence which differsfrom SEQ ID No. 4 or SEQ ID No. 5 or SEQ ID No. 6 or SEQ ID No. 12 orSEQ ID No. 13 or SEQ ID No. 14 or SEQ ID No. 16 or SEQ ID No. 17 or SEQID No. 18 due to the degeneracy of the genetic code.

A further aspect of the present invention provides, a method ofproducing a fermented beverage (e.g. beer) comprising the steps of: (a)preparing a mash, (b) filtering the mash to obtain a wort, and (c)fermenting the wort to obtain a fermented beverage, such as a beer,wherein xylanase comprising (or consisting of) a polypeptide sequenceshown herein as SEQ ID No. 1, SEQ ID No. 2, SEQ ID No. 3, SEQ ID No. 9,SEQ ID No. 10, SEQ ID No. 11 or SEQ ID No. 15, or a variant, homologue,fragment or derivative thereof having at least 75% identity (such as atleast 80%, 85%, 90%, 95%, 98% or 99% identity) with SEQ ID No. 1 or SEQID No. 2, SEQ ID No. 3, SEQ ID No. 9, SEQ ID No. 10, SEQ ID No. 11 orSEQ ID No. 15; or a polypeptide sequence which comprises SEQ ID No. 1,SEQ ID No. 2, SEQ ID No. 3, SEQ ID No. 9, SEQ ID No. 10, SEQ ID No. 11or SEQ ID No. 15 with a conservative substitution of at least one of theamino acids; or a xylanase which is encoded by a nucleotide sequenceshown herein as SEQ ID No. 4, SEQ ID No. 5, SEQ ID No. 6, SEQ ID No. 12,SEQ ID No. 13, SEQ ID No. 14, SEQ ID No. 16; SEQ ID No. 17 or SEQ ID No.18, or a nucleotide sequence which can hybridize to SEQ ID No. 4, SEQ IDNo. 5, SEQ ID No. 6, SEQ ID No. 12, SEQ ID No. 13, SEQ ID No. 14, SEQ IDNo. 16, SEQ ID No. 17 or SEQ ID No. 18 under high stringency conditions,or a nucleotide sequence which has at least 75% identity (such as atleast 80%, 85%, 90%, 95% or 98% identity) with SEQ ID No. 4, SEQ ID No.5, SEQ ID No. 6, SEQ ID No. 12, SEQ ID No. 13, SEQ ID No. 14, SEQ ID No.16, SEQ ID No. 17 or SEQ ID No. 18; or a nucleotide sequence whichdiffers from SEQ ID No. 4 or SEQ ID No. 5 or SEQ ID No. 6 or SEQ ID No.12 or SEQ ID No. 13 or SEQ ID No. 14 or SEQ ID No. 16 or SEQ ID No. 17or SEQ ID No. 18 due to the degeneracy of the genetic code, is added to:(i) the mash of step (a) and/or (ii) the wort of step (b) and/or (iii)the wort of step (c).

The present invention yet further provides a fermented beverage, such asa beer, produced by a method of present invention.

For the avoidance of doubt, SEQ ID No. 3 is the mature form of SEQ IDNo. 1 or SEQ ID No. 2.

Likewise, SEQ ID No. 11 is the mature form of SEQ ID No. 9 or SEQ ID No10.

Detailed Disclosure of the Preferred Embodiments of the Invention

Unless defined otherwise, all technical and scientific terms used hereinhave the same meaning as commonly understood by one of ordinary skill inthe art to which this disclosure belongs. Singleton, et al, DICTIONARYOF MICROBIOLOGY AND MOLECULAR BIOLOGY, 20 ED., John Wiley and Sons, NewYork (1994), and Hale & Marham, THE HARPER COLLINS DICTIONARY OFBIOLOGY, Harper Perennial, NY (1991) provide one of skill with a generaldictionary of many of the terms used in this disclosure.

This disclosure is not limited by the exemplary methods and materialsdisclosed herein, and any methods and materials similar or equivalent tothose described herein can be used in the practice or testing ofembodiments of this disclosure. Numeric ranges are inclusive of thenumbers defining the range. Unless otherwise indicated, any nucleic acidsequences are written left to right in 5′ to 3′ orientation; amino acidsequences are written left to right in amino to carboxy orientation,respectively.

The headings provided herein are not limitations of the various aspectsor embodiments of this disclosure which can be had by reference to thespecification as a whole. Accordingly, the terms defined immediatelybelow are more fully defined by reference to the specification as awhole.

Amino acids are referred to herein using the name of the amino acid, thethree letter abbreviation or the single letter abbreviation.

The term “protein”, as used herein, includes proteins, polypeptides, andpeptides.

As used herein, the term “amino acid sequence” is synonymous with theterm “polypeptide” and/or the term “protein”. In some instances, theterm “amino acid sequence” is synonymous with the term “peptide”. Insome instances, the term “amino acid sequence” is synonymous with theterm “enzyme”.

The terms “protein” and “polypeptide” are used interchangeably herein.In the present disclosure and claims, the conventional one-letter andthree-letter codes for amino acid residues may be used. The 3-lettercode for amino acids as defined in conformity with the IUPACIUB JointCommission on Biochemical Nomenclature (JCBN). It is also understoodthat a polypeptide may be coded for by more than one nucleotide sequencedue to the degeneracy of the genetic code.

Other definitions of terms may appear throughout the specification.Before the exemplary embodiments are described in more detail, it is tounderstand that this disclosure is not limited to particular embodimentsdescribed, as such may, of course, vary. It is also to be understoodthat the terminology used herein is for the purpose of describingparticular embodiments only, and is not intended to be limiting, sincethe scope of the present disclosure will be limited only by the appendedclaims.

Where a range of values is provided, it is understood that eachintervening value, to the tenth of the unit of the lower limit unlessthe context clearly dictates otherwise, between the upper and lowerlimits of that range is also specifically disclosed. Each smaller rangebetween any stated value or intervening value in a stated range and anyother stated or intervening value in that stated range is encompassedwithin this disclosure. The upper and lower limits of these smallerranges may independently be included or excluded in the range, and eachrange where either, neither or both limits are included in the smallerranges is also encompassed within this disclosure, subject to anyspecifically excluded limit in the stated range. Where the stated rangeincludes one or both of the limits, ranges excluding either or both ofthose included limits are also included in this disclosure.

It must be noted that as used herein and in the appended claims, thesingular forms “a”, “an”, and “the” include plural referents unless thecontext clearly dictates otherwise. Thus, for example, reference to “anenzyme” includes a plurality of such candidate agents and reference to“the feed” includes reference to one or more feeds and equivalentsthereof known to those skilled in the art, and so forth.

The publications discussed herein are provided solely for theirdisclosure prior to the filing date of the present application. Nothingherein is to be construed as an admission that such publicationsconstitute prior art to the claims appended hereto.

Increasing prices of raw material traditionally used as energy source inanimal feed, as a feedstock in biofuel production, as an ingredient inbrewing or malting, or as a feedstock in wheat gluten-starch separationprocesses for instance have resulted in inclusion of low-cost fibrousmaterials in the starting substrates for these industries, particularlythe use of low-cost fibrous by-products in animal feed.

Fibre addition may cause several disadvantageous effects. For example inanimal feed fibre addition may cause anti-nutritional effects. Thepresence of un-degraded polymers present in the animal's intestinecauses a highly viscous content and, impeded diffusion with reducednutrient absorption as a result. Also, the polymers possess a high waterholding capacity hindering an effective re-absorption of water, and thewater retention increases the volume of the gut content, which leads toa decrease intestinal transit time (Englyst & Kingman (1993) in HumanNutrition and Dietetics, 9th edition (Garrow J. S., James W. P. T.,eds.) p. 53).

In feedstuffs, hemicellulose and cellulose (including insolublearabinoxylan) also form physical barriers encapsulating (or entrapping)nutrients like starch and protein and thereby retaining access to thesenutrients for the animal.

Hemicellulose and cellulose (including insoluble arabinoxylans(AXinsol)) by themselves are also potential energy sources, as theyconsist of C5- and C6-saccharides. Mono C6-saccharides can be used asenergy source by the animal, while oligo C5-saccharides can betransformed into short chain fatty acids by the micro flora present inthe animal gut (van den Broek et al., 2008 Molecular Nutrition & FoodResearch, 52, 146-63), which short chain fatty acids can be taken up anddigested by the animal's gut.

Release of nutrients and water from feedstuffs as a consequence ofphysical barrier degradation is dependent on the ability of the xylanaseto degrade insoluble fibre components (e.g. Insoluble arabanoxylans(AXinsol)).

In one aspect, the present invention relates to a novel xylanase.

In one embodiment the present invention provides a xylanase enzymecomprising (or consisting of) a polypeptide sequence shown herein as SEQID No. 1, SEQ ID No. 2 or SEQ ID No. 3 or a variant, homologue, fragmentor derivative thereof having at least 98.4% (e.g. at least 98.5 or 98.8or 99 or 99.1 or 99.5%) identity with SEQ ID No. 1 or SEQ ID No. 2 orSEQ ID No. 3.

The present invention also relates to a xylanase which is encoded by anucleotide sequence shown herein as SEQ ID No. 4, SEQ ID No. 5 or SEQ IDNo. 6, or a nucleotide sequence which can hybridize to SEQ ID No. 4, SEQID No. 5 or SEQ ID No. 6 under high stringency conditions, or anucleotide sequence which has at least 97.7% (e.g. at least 98%, 98.5%or 99%) identity with SEQ ID No. 4, SEQ ID No. 5 or SEQ ID No. 6.

In one aspect, the xylanase enzyme of the present invention may beobtainable from (or obtained from) a fungus, namely Fusariumverticilloides.

In another aspect, the present invention provides a xylanase obtainablefrom (or obtained from) Fusarium verticilloides for use in feed or afeed additive composition.

In one embodiment the xylanase enzyme of the present invention may bereferred to herein as FveXyn4 or Hifi168.

The present invention yet further provides a nucleic acid comprising (orconsisting of) a nucleotide sequence shown herein as SEQ ID No. 4, SEQID No. 5 or SEQ ID No. 6; or a nucleotide sequence which has at least97.7% (e.g. at least 98%, 98.5% or 99%) identity with SEQ ID No. 4, SEQID No. 5 or SEQ ID No. 6.

Both the polypeptide sequences and the nucleic acid sequences taughtherein are preferably isolated.

The xylanase of the present invention is preferably a GH10 xylanase. Inother words the xylanase may have a molecular weight in the range of32-39 kDa and/or the catalytic domain of the xylanase consists of aneightfold β/α barrel structure (as taught in Harris et al 1996—Acta.Crystallog. Sec. D 52, 393-401).

In one aspect, the present invention provides a composition comprising axylanase enzyme comprising (or consisting of) a polypeptide sequenceshown herein as SEQ ID No. 1, SEQ ID No. 2 or SEQ ID No. 3 or a variant,homologue, fragment or derivatives thereof having at least 98.5% (e.g.at least 98.8 or 99 or 99.1 or 99.5%) identity with SEQ ID No. 1 or SEQID No. 2 or SEQ ID No. 3 or a xylanase which is encoded by a nucleotidesequence shown herein as SEQ ID No. 4, SEQ ID No. 5 or SEQ ID No. 6, ora nucleotide sequence which has at least 97.7% (e.g. at least 98%, 98.5%or 99%) identity with SEQ ID No. 4, SEQ ID No. 5 or SEQ ID No. 6.

In one aspect, the xylanase enzyme for use in the methods and uses ofthe present invention comprises (or consists of) a polypeptide sequenceshown herein as SEQ ID No. 1, SEQ ID No. 2, SEQ ID No. 3, SEQ ID No. 9,SEQ ID No. 10, SEQ ID No. 11, or SEQ ID No. 15 or a variant, homologue,fragment or derivative thereof having at least 75% (suitably at least85% or at least 90% or at least 99%) identity with SEQ ID No. 1 or SEQID No. 2 or SEQ ID No. 3 or SEQ ID No. 9 or SEQ ID No. 10 or SEQ ID No.11 or SEQ ID No. 15, or a polypeptide sequence which comprises SEQ IDNo. 1, SEQ ID No. 2, SEQ ID No. 3, SEQ ID No. 9, SEQ ID No. 10, SEQ IDNo. 11 or SEQ ID No. 15 with a conservative substitution of at least oneof the amino acids; or a xylanase which is encoded by a nucleotidesequence shown herein as SEQ ID No. 4, SEQ ID No. 5, SEQ ID No. 6, SEQID No. 12, SEQ ID No. 13, SEQ ID No. 14, SEQ ID No. 16, SEQ ID No. 17 orSEQ ID No. 18, of a nucleotide sequence which has at least 75% (suitablyat least 85%, or at least 90% or at least 98%) identity with SEQ ID No.4, SEQ ID No. 5, SEQ ID No. 6, SEQ ID No. 12, SEQ ID No. 13, SEQ ID No.14, SEQ ID No. 16, SEQ ID No. 17 or SEQ ID No. 18, or a nucleotidesequence which differs from SEQ ID No. 4 or SEQ ID No. 5 or SEQ ID No. 6or SEQ ID No. 12 or SEQ ID No. 13 or SEQ ID No. 14 or SEQ ID No. 16 orSEQ ID No. 17 or SEQ ID No. 18 due to the degeneracy of the geneticcode.

In one aspect, the xylanase enzyme for use in the methods and uses ofthe present invention comprises (or consists of) a polypeptide sequenceshown herein as SEQ ID No. 1, SEQ ID No. 2, SEQ ID No. 3, SEQ ID No. 9,SEQ ID No. 10, SEQ ID No. 11 or SEQ ID No. 15 or a variant, homologue,fragment or derivative thereof having at least 85% identity with SEQ: IDNo. 1 or SEQ ID No. 2 or SEQ ID No. 3 or SEQ ID No. 9 or SEQ ID No. 10or SEQ ID No. 11 or SEQ ID No. 15; or a polypeptide sequence whichcomprises SEQ ID No. 1, SEQ ID No. 2, SEQ ID No. 3, SEQ ID No. 9, SEQ IDNo. 10, SEQ ID No. 11 or SEQ ID No. 15 with a conservative substitutionof at least one of the amino acids; or a xylanase which is encoded by anucleotide sequence shown herein as SEQ ID No. 4, SEQ ID No. 5, SEQ IDNo. 6, SEQ ID No. 12, SEQ ID No. 13, SEQ ID No. 14, SEQ ID No. 16, SEQID No. 17 or SEQ ID No. 18 or a nucleotide sequence which has at least85% identity with SEQ ID No. 4, SEQ ID No. 5, SEQ ID No. 6, SEQ ID No.12, SEQ ID No. 13, SEQ ID No. 14, SEQ ID No. 16, SEQ ID No. 17 or SEQ IDNo. 18, or a nucleotide sequence which differs from SEQ ID No. 4 or SEQID No. 5 or SEQ ID No. 6 or SEQ ID No. 12 or SEQ ID No. 13 or SEQ ID No.14 or SEQ ID No. 16 or SEQ ID No. 17 or SEQ ID No. 18 due to thedegeneracy of the genetic code.

In one aspect, the xylanase enzyme for use in the methods and uses ofthe present invention comprises (or consists of) a polypeptide sequenceshown herein as SEQ ID No. 1, SEQ ID No. 2, SEQ ID No. 3, SEQ ID No. 9,SEQ ID No. 10, SEQ ID No. 11 or SEQ ID No. 15 or a variant, homologue,fragment or derivative thereof having at least 90% identity with SEQ IDNo. 1 or SEQ ID No. 2 or SEQ ID No. 3 or SEQ ID No. 9 or SEQ ID No. 10or SEQ ID No. 11 or SEQ ID No. 15; or a polypeptide sequence whichcomprises SEQ ID No. 1, SEQ ID No. 2, SEQ ID No. 3, SEQ ID No. 9, SEQ IDNo. 10, SEQ ID No. 11 or SEQ ID No. 15 with a conservative substitutionof at least one of the amino acids; or a xylanase which is encoded by anucleotide sequence shown herein as SEQ ID No. 4, SEQ ID No. 5, SEQ IDNo. 6, SEQ ID No. 12, SEQ ID No. 13, SEQ ID No. 14, SEQ ID No. 16, SEQID No. 17 or SEQ ID No. 18 or a nucleotide sequence which has at least90% identity with SEQ ID No. 4, SEQ ID No. 5, SEQ ID No. 6, SEQ ID No.12, SEQ ID No. 13, SEQ ID No. 14, SEQ ID No. 16, SEQ ID No. 17 or SEQ IDNo. 18, or a nucleotide sequence which differs from SEQ ID No. 4 or SEQID No. 5 or SEQ ID No. 6 or SEQ ID No. 12 or SEQ ID No. 13 or SEQ ID No.14 or SEQ ID No. 16 or SEQ ID No. 17 or SEQ ID No. 18 due to thedegeneracy of the genetic code.

In one aspect, the xylanase enzyme for use in the methods and uses ofthe present invention comprises (or consists of) a polypeptide sequenceshown herein as SEQ ID No. 1, SEQ ID No. 2, SEQ ID No. 3, SEQ ID No. 9,SEQ ID No. 10, SEQ ID No. 11 or SEQ ID No. 15 or a variant, homologue,fragment or derivative thereof having at least 99% identity with SEQ IDNo. 1 or SEQ ID No. 2 or SEQ ID No. 3 or SEQ ID No. 9 or SEQ ID No. 10or SEQ ID No. 11 or SEQ ID No. 15, or a polypeptide sequence whichcomprises SEQ ID No. 1, SEQ ID No. 2, SEQ ID No. 3, SEQ ID No. 9, SEQ IDNo. 10, SEQ ID No. 11 or SEQ ID No. 15 with a conservative substitutionof at least one of the amino acids; or a xylanase which is encoded by anucleotide sequence shown herein as SEQ ID No. 4, SEQ ID No. 5, SEQ IDNo. 6, SEQ ID No. 12, SEQ ID No. 13, SEQ ID No. 14, SEQ ID No. 16, SEQID No. 17 or SEQ ID No. 18, or a nucleotide sequence which has at least98% identity with SEQ ID No. 4, SEQ ID No. 5, SEQ ID No. 6, SEQ ID No.12, SEQ ID No. 13, SEQ ID No. 14, SEQ ID No. 16, SEQ ID No. 17 or SEQ IDNo. 18, or a nucleotide sequence which differs from SEQ ID No. 4 or SEQID No. 5 or SEQ ID No. 6 or SEQ ID No. 12 or SEQ ID No. 13 or SEQ ID No.14 or SEQ ID No. 16 or SEQ ID No. 17 or SEQ ID No. 18 due to thedegeneracy of the genetic code.

In one embodiment the xylanase enzyme of the present invention may bereferred to herein as FveXyn4 (this term refers to the active protein,e.g. the mature protein).

In one embodiment the xylanase enzyme of the present invention may bereferred to herein as FoxXyn2 (this term refers to the active protein,e.g. the mature protein).

In one embodiment preferably the xylanase is a fungal xylanase.

In one embodiment preferably the xylanase is a GH10 xylanase.

In one embodiment preferably the xylanase is a fungal GH10 xylanase.

In one embodiment preferably the xylanase in an endoxylanase, e.g. anendo-1,4-β-d-xylanase. The classification for an endo-1,4-β-d-xylanaseis E.C. 3.2.1.8.

Preferably the xylanase of the present invention has an optimum pH atabout 6.

Preferably the xylanase of the present invention retains greater than70% of maximum activity between pH 4.6 and 7, preferably between 5.1 and7.

Without wishing to be bound by theory, pH may also have an importanteffect on enzyme efficacy and efficiency. For feed applications inparticular the pH profile of the xylanases of the present inventionfavour activity in the small intestine, under neutral conditions.

Preferably the xylanase of the present invention has an optimumtemperature of about 60° C.

Preferably the xylanase of the present invention retains greater than70% of maximum activity between 45° C. and 64° C.

In one embodiment, the xylanases according to the present invention iscapable of degrading (or degrades) a xylan-containing material,particularly arabinoxylans, particularly isoluble arabinoxylans(AXinsol).

In another embodiment the xylanases according to the present inventionis capable of degrading (or degrades) soluble polymers (e.g. oligomers)that are produced from degradation of AXinsol or that are (naturally)present in grain-based material.

In a further embodiment the xylanases according the present invention iscapable of degrading (or degrades) both a xylan-containing material,particularly arabinoxylans, particularly AXinsol, and soluble polymers(e.g. oligomers) that are produced from degradation of AXinsol.

In one embodiment the xylanases of the present invention are unaffectedby wheat xylanases inhibitors, e.g. proteinaceous inhibitors, e.g.TAXI-like proteinaceous inhibitors in wheat. Prior art fungal xylanasescan be inhibited by as much as 70-95% by wheat proteinaceous inhibitors.Preferably the xylanases of the present invention are only inhibited by20-30% at most in wheat applications.

TAXI are Triticum aestivum xylanases inhibitors, present in cereals.

The term “consisting essentially of” as used herein means thatunspecified components may be present if the characteristics of theclaimed composition are thereby not materially affected.

The term “consisting of” means that the proportions of the specificingredients must total 100%.

The term “comprising” used herein may be amended in some embodiments torefer to consisting essentially of or consisting of (both having a morelimited meaning that “comprising”).

In one embodiment the insoluble arabinoxylan containing material is notwheat straw.

Uses

The xylanase of the present invention can be suitably used in any one ofthe following applications:

a) An additive in animal feedstuffs; and/or

b) A feed supplement for an animal; and/or

c) Breakdown of grain-based material (e.g. this can be whole grain orpart of grain). The breakdown products (e.g. glucose) can be used as afeedstock for any fermentation process, such as in biofuel (e.g.bioethanol) production or in the production of other products such asbiochemicals (e.g., bio-based isoprene). Therefore in one embodiment thepresent invention relates to the production of biofuel (e.g. bioethanol)and to the enhanced utilisation of grain-based material in the biofuelindustry; and/or

c) Cereal (e.g. wheat) gluten-starch separation industry. The resultantproduct(s) may be starch (e.g. purified starch) and/or gluten and/orfibres and/or water solubles (such as soluble pentosans). In oneembodiment the present invention relates to the production of starchand/or gluten; and/or

d) Improving malting and brewing, e.g. by breaking down grain-basedmaterial (e.g. malted barley).

In one embodiment the xylanase of the present invention is used in afeedstuff. Preferably a feedstuff comprising corn or is a corn-basedfeedstuff.

In one embodiment the xylanase of the present invention is used inmalting or brewing.

In a further embodiment the xylanase of the present invention is used inwheat gluten-starch separation.

In a yet further embodiment the xylanase of the present invention isused in the breakdown of grain-based material and may be part of thebiofuel (e.g. bioethanol) production process.

For the avoidance of doubt, SEQ ID No. 3 is the mature form of SEQ IDNo. 1 or SEQ ID No. 2.

Likewise, SEQ ID No. 11 is the mature form of SEQ ID No. 9 or SEQ ID No.10.

Advantages

The novel xylanase taught herein has many advantages compared with knownxylanases.

The xylanases as taught herein and of the present invention areunexpectedly good at solubilising pentosans.

The xylanases as taught herein and of the present invention areunexpectedly good at solubilising AXinsol.

Surprisingly it has been found that the xylanase of the presentinvention is particularly good at degrading xylan-containing materials,such as arabinoxylans, e.g. AXinsol, in a broad spectrum of substrates,corn, wheat, DDGS, etc, in particular corn and corn based substrates, inparticular both wheat (including wheat-based) products and corn(including corn-based products). Compared with the benchmark xylanaseswhich are all commercially produced and marketed xylanases, the novelxylanase taught herein was capable of much more efficient degradationand pentosan release from more plant based materials (in particularcorn-based substrates) compared with the marketed xylanases. This wascompletely unexpected. This contrasts with prior-known enzymes, whichare often inferior at solubilising AXinsol in corn or corn-basedsubstrates of which are not as efficient in both wheat- and corn-basedsubstrates.

In addition, the enzyme of the present invention is particularly good atnot only breaking down (solubilising) AXinsol, but also breaking down(or degrading) the solubilized polymers efficiently. By being able toefficiently (quickly) breakdown (degrade) the solubilized polymers(obtained from dissolving AXinsol) a reduction in viscosity is obtained.This latter effect is essential in some of the claimed applications.

Typically, conventional xylanases may breakdown AXinsol, but will leadto an increase is the polymer production products which will lead to anincrease in viscosity of the mixture. This increased viscosity isdisadvantageous in many applications.

The enzymes of the present invention and as described herein have beenfound to not only breakdown (solubilise) insoluble arabinoxylans(AXinsol) from a wide range of substrates, including corn, wheat, DDGS,etc, in particular corn and corn-based substrates, in particular bothwheat (including wheat-based) products and corn (including corn-basedproducts), but also efficiently breakdown the thus solubilised polymersto ensure viscosity is not raised and/or to reduce viscosity.

The xylanases of the present invention and as described herein arecapable of degrading AXsol or the breakdown products of AXinsol toensure viscosity is not increased and/or viscosity is reduced in thereaction mixture.

Many of the xylanases commercialized for use in feedstuffs forsolubilizing pentosans are GH11 enzymes. It had been considered by thoseskilled in the art that GH10 xylanases were not as strong atsolubilizing pentosans, particularly AXinsol, compared with GH11xylanases. Surprisingly it has been found that the novel xylanasedisclosed herein which is a GH10 xylanase is particularly good atsolubilizing AXinsol in a broad spectrum of substrates, including cornbased substrates. Surprisingly, the present inventors have found thatthe GH10 xylanases of the present invention (and taught herein)outperform commercial GH11 xylanases in their ability to solubilizepentosans.

The fact that the present enzymes efficiently solubilize AXinsol fromcorn and corn-based substrates is significantly advantageous as cornholds much more AX in the insoluble form compared with other cereals,such as wheat and rye for example. Therefore only xylanases that canbreakdown AXinsol can show significant benefit to animals fed oncorn-soy diet for example.

It was completely unexpected for a GH10 xylanase to be so good onsolubilizing AXinsol in cereals, particularly in corn or corn-basedsubstrates.

The enzymes of the present invention are able to efficiently (andquickly) degrade the polymers and/or oligomers that are produced fromsolubilisation of AXinsol or that are present in grain-based materials.This leads to an unexpected advantage for the GH10 xylanases taughtherein in that they are particularly good, in a number of applicationsto keep viscosity low or to reduce viscosity, e.g. in feedstuffs; inbrewing and/or malting; in grain-based production of glucose, e.g. forfurther processing to biofuels and/or biochemicals (e.g., bio-basedisoprene); or in the wheat gluten-starch separation industry for theproduction of starch for example.

Notably it has been found that the degradation product on average isshorter for the GH10 enzymes tested herein compared with GH11 enzymes.This enhances the lowering of viscosity effect.

In addition, a further advantage of the the GH10 xylanases of thepresent invention (unlike many GH11 xylanases) are unaffected by wheatxylanase inhibitors, e.g., TAXI like proteinaceous inhibitors, whichoccur in wheat.

One advantage of the present invention is that it improves wheatgluten-starch separation.

The enzyme of the present invention is particularly effective atenhancing the performance of a subject or improving the digestibility ofa raw material in a feed and/or for improving feed efficiency in asubject.

Xylan-Containing Material

The xylanase of the present invention (or composition comprising thexylanase of the present invention) may be used to degrade anyxylan-containing material.

In one embodiment the xylan-containing material is any plant materialcomprising arabinoxylan.

In one embodiment the xylan-containing material is any plant materialcomprising insoluble arabinoxylan (AXinsol).

In one embodiment the xylan-containing material is a feedstuff or feedcomponent.

In one embodiment the xylan-containing material is a grain-basedmaterial (including whole grains or partial grains or malted grains,e.g. malted barley). When the method relates to biofuel production (e.g.bioethanol production) then preferably the xylan-containing material isa grain-based material.

In another embodiment the xylan-containing material may be a barley maltor mash, or malted barley or combinations thereof.

In a yet further embodiment the xylan-containing material may be acereal flour (e.g. wheat, oat, rye or barley flour). When the methodrelates to a gluten-starch separation process preferably thexylan-containing material is a cereal flour (e.g. wheat oat, rye orbarley flour).

Breakdown or Degradation

The enzyme (or composition comprising the enzyme) of the presentinvention or as disclosed herein may be used to breakdown (degrade)AXinsol or AXsol or degradation products of AXinsol.

The term “breakdown” or “degrade” in synonymous with hydrolyses.

Solubilisation/Degradation

The present invention relates to a method of degrading axylan-containing material (preferably an arabinoxylan-containingmaterial, preferably an insoluble arabinoxylan (AXinsol)-containingmaterial) to produce soluble pentosans (which can be polymeric,oligomeric or monomeric).

This method may be described herein as pentosan solubilisation orarabinoxylan solubilisation or AXinsol solubilisation or degradation ofAXinsol.

In one embodiment, the present invention relates to a method ofdegrading (or breaking down) insoluble arabinoxylan (AXinsol). This canalso be referred to as solubilisation of insoluble arabinoxylan and/orsolubilisation of pentosans.

In a further embodiment of the present invention the method relates todegrading (e.g. breaking down) polymers derived from the degradation ofinsoluble arabinoxylans.

Arabinoxylan (AX)

The term “arabinoxylans” (AX) as used herein means a polysaccharideconsisting of a xylan backbone (1,4-linked xylose units) withL-arabinofuranose (L-arabinose in its 5-atom ring form) attachedrandomly by 1α→2 and/or 1α→3 linkages to the xylose units throughout thechain. Arabinoxylan is a hemicellulose found in both the primary andsecondary cell walls of plants. Arabinoxylan can be found in the bran ofgrains such as wheat, maize (corn), rye, and barley.

Arabinoxylan (AX) is found in close association with the plant cellwall, where it acts as a glue linking various building blocks of theplant bell wall and tissue, give it both structural strength andrigidity.

The term “pentosan” as used herein is any of a group of carbohydrateswhich yield pentoses on complete hydrolysis.

Since xylose and arabinose (the constituents of arabinoxylans) are bothpentoses, arabinoxylans are usually classified as pentosans.

AX is the principal Non Starch Polysaccharide (NSP)-fraction in severalof the most important feed raw material, including wheat and corn.

Its abundance, location within vegetable material and molecularstructure cause AX to have a severe, negative impact on feeddigestibility, effectively reducing the nutritional value of the rawmaterials in which it is present. This makes AX an importantanti-nutritional factor, reducing animal production efficiency.

In addition AX can have a severe, negative impact when trying tobreakdown plant material for example in processes such as brewing,malting, biofuel manufacture, effectively reducing the amount ofsubstrate accessible in the raw plant material.

AXs can also hold substantial amounts of water (which can be referred toas their water holding capacity)—this can cause soluble arabinoxylans toresult in (high) viscosity—which is a disadvantage in many applications.

The term “Hemicellulose”—as used herein means the polysaccharidecomponents of plant cell walls other than cellulose. The term“hemicellulose” as used herein may mean polysaccharides in plant cellwalls which are extractable by dilute alkaline solutions. Hemicellulosescomprise almost one-third of the carbohydrates in woody plant tissue.The chemical structure of hemicelluloses consists of long chains of avariety of pentoses, hexoses, and their corresponding uronic acids.Hemicelluloses may be found in fruit, plant stems, and grain hulls.Xylan is an example of a pentosan consisting of D-xylose units with 1β→4linkages.

Water Insoluble Arabinoxylan (AXinsol)

Water-insoluble arabinoxylan (AXinsol) also known as water-unextractablearabinoxylan (WU-AX) constitutes a significant proportion of the drymatter of plant material.

In wheat AXinsol can account for 6.3% of the dry matter. In wheat branand wheat DDGS AXinsol can account for about 20.8% or 13.4% of the drymatter (w/w).

In rye AXinsol can account for 5.5% of the dry matter.

In corn AXinsol can account for 3.5-6% (e.g. 5.1%) of the dry matter. Incorn DDGS AXinsol can account for 10-20% (e.g. 12.6%) of the dry matter.

AXinsol causes nutrient entrapment in feed. Large quantities of welldigestible nutrients such as starch and proteins remain either enclosedin clusters of cell wall material or bound to side chains of the AX.These entrapped nutrients will not be available for digestion andsubsequent absorption in the small intestine.

Water-Soluble Arabinoxylan (AXsol)

Water-soluble arabinoxylan (AXsol) also known as water extractablearabinoxylan (WE-AX) can cause problems in biofuel production and/ormalting and/or brewing and/or in feed as they can cause increasedviscosity due to the water-binding capacity of AXsol.

In feed AXsol can have an anti-nutritional effect particularly inmonogastrics as they cause a considerable increase of the viscosity ofthe intestinal content, caused by the extraordinary water-bindingcapacity of AXsol. The increase viscosity can affect feed digestion andnutrient use as it can prevent proper mixing of feed with digestiveenzymes and bile salts and/or it slows down nutrient availability andabsorption and/or it stimulates fermentation in the hindgut.

In wheat AXsol can account for 1.8% of the dry matter. In wheat bran andwheat DDGS AXsol can account for about 1.1% or 4.9% of the dry matter(w/w).

In rye AXsol can account for 3.4% of the dry matter.

In barley AXsol can account for 0.4-0.8% of the dry matter.

In corn AXsol can account for 0.1-0.4% (e.g. 0.1%) of the dry matter. Incorn DDGS AXinsol can account for 0.3-2.5% (e.g. 0.4%) of the drymatter.

In addition, however, to the amount of AXsol present in plant material,when a xylanase solubilises AXinsol in the plant material this canrelease pentosans and/or oligomers which contribute to AXsol content ofthe plant material.

One significant advantage of the xylanases disclosed herein is that theyhave the ability to solubilise AXinsol without increasing viscosity. Itis presently believed that high molecular weight products are not formed

A breakdown of AXsol can decrease viscosity.

A breakdown of AXsol can release nutrients.

Viscosity

The present invention can be used to ensure that the viscosity is notincreased and/or to reduce viscosity in any process where thewater-binding capacity of AXsol causes an undesirable increase inviscosity.

The present invention relates to ensuring that viscosity is notincreased and/or to reducing viscosity by breaking down (degrading)AXsol or by breaking down (degrading) the polymers and/or oligomersproduced by solubilising AXinsol.

Without wishing to be bound by theory, by being able to efficiently(quickly) breakdown (degrade) the solubilized polymers (e.g. oligomers)obtained from dissolving AXinsol an undesirable increase in viscositycan be avoided and/or a reduction in viscosity can be obtained. The term“efficiently” as used herein means that the enzyme is capable ofdegrading the polymers (e.g. oligomers) being formed by solubilisationof the AXinsol faster than the speed with which the AXinsol is degraded(or solubilized).

Reducing viscosity has advantages in many applications as taught herein.

An in vitro assay which attempts to mimic the environment in the smallintestine of a chicken was originally described by Bedford & Classen(1993 Poultry Sci., 72, 137-143). The assay consists of a two stepsincubation of the feed first at low pH with pepsin followed byincubation with pancreatin at neutral pH. It is generally accepted thatthe viscosity of the supernatant after end incubation correlates withthe viscosity created in vivo in broilers.

Without increasing viscosity and/or a reduction in viscosity as taughtherein for feed applications means that addition of the xylanase willresult In an unchanged or lower viscosity measured by the methoddescribed in Example 7. By unchanged it is meant that the measuredvalue, being the average of three repetitions, falls within two standarddeviation of the measured value for a wheat sample without xylanaseaddition.

Viscosity can be measured using the following devices: RapidViscoAnalyzer (RVA) (e.g. in bioethanol processing) and Haake VT550viscometer (Thermofisher) (e.g. is wheat-gluten starch processing). Bothdevices can monitor viscosity profiles of fuel ethanol processes andwheat starch separation processes, of which the experimental conditionsare taught in Example 8 and 9, respectively.

In the present invention a reduction in viscosity can be calculated bycomparing one sample comprising the xylanase of the present invention(or taught herein) compared with another comparable sample without thexylanase of the present invention (or taught herein).

Comparing the viscosity reduction profiles of the xylanase of thepresent Invention with those of the market benchmark xylanase(s)demonstrates the enzyme performance. The aim is to improve enzymeperformance compared to the market benchmark. The benchmark enzyme(s)for the individual applications are provided in the examples below.

The benchmark enzyme for feed applications Is Econase®XT.

The benchmark enzyme for bioethanol processing is Xylathin™.

The benchmark enzyme for wheat-gluten starch separation is ShearzymePlus™.

In one embodiment of the present invention the xylanases taught hereinare viscosity reducers.

Generally, wheat (or other cereal) is first dry-milled to separate thebran and germ from the endosperm, which is ground into flour. Thisendosperm flour is then further fractionated through a wheat starchseparation process into several product streams of varying commercialvalue. The major aim is to produce a refined grade of A-starch,consisting of large, lenticular granules of 15-40 μm. The second streamB-starch consists of less purified starch granules, which are sphericaland small (1-10 μm). (C. C. Maningat, P. A. Selb, S. D. Bassi, K. S.Woo, G. D. Lasater, Chapter 10 from the book “Starch” (2009) 441-451,Wheat starch: production, properties, modification and uses). Isolatedwheat starch forms the starting material for modified starch productionwith applications in both food- and nonfood-applications. Vital glutenis the third product of added-value in wheat separation processes. Thevitality of the isolated wheat gluten is determined by the ability toform viscoelastic networks, required for breadmaking. Vital glutenencapsulate the carbon dioxide formed in dough preparation duringbaking, and consequently increase the bread volume. (Anne van derBorght, Hans Goesaert, Wim S. Veraverbeke, Jan A. Delcour, Journal ofCereal Science 41 (2005) 221-237, Fractionation of wheat and wheat flourinto starch and gluten: overview of the main processes and the factorsinvolved.) It is therefore often used to enrich flours for bread making,to achieve improved bread products. Other markets for gluten include asan additive in vegetarian, meat, fish or poultry products, includingthose in pet-food industry; in cereal breakfast; or in soy sauce. Due toits thermoplasticity and good film-forming properties, gluten is alsoused in non-food markets as adhesives. (L. Day, M. A. Augustin, I. L.Batey, C. W. Wrigley, Trends in Food Science & Technology 17 (2006)82-90, Wheat-gluten uses and industry needs.).

The xylanases taught herein can be used to reduce the viscosity (or notincrease viscosity) in processes for separating cereal flour (e.g.wheat, oat, rye or barley flour) into starch and gluten fractions and toimprove the separation by degrading oligosaccharides that hinder glutenagglomeration.

Wort viscosity, and the viscosity of barley mash and barley malt inbrewing and malting can cause significant disadvantages during brewingand/or malting. The present invention relates to reducing the viscosity(or not increase the viscosity) of wort, barley mash, barley malt or acombination thereof.

Feed or Feedstuff

The enzyme or feed additive composition of the present invention may beused as—or in the preparation of—a feed.

The term “feed” is used synonymously herein with “feedstuff”.

Preferably the arabinoxylan-containing material of the present inventionis a feedstuff, or a constituent of a feedstuff, or a feed component.

The feed may be in the form of a solution or as a solid or as asemi-solid—depending on the use and/or the mode of application and/orthe mode of administration.

When used as—or in the preparation of—a feed—such as functional feed—theenzyme or composition of the present invention may be used inconjunction with one or more of: a nutritionally acceptable carrier, anutritionally acceptable diluent, a nutritionally acceptable excipient,a nutritionally acceptable adjuvant, a nutritionally active ingredient.

In a preferred embodiment the enzyme or feed additive composition of thepresent invention is admixed with a feed component to form a feedstuff.

The term “feed component” as used herein means all or part of thefeedstuff. Part of the feedstuff may mean one constituent of thefeedstuff or more than one constituent of the feedstuff, e.g. 2 or 3 or4. In one embodiment the term “feed component” encompasses a premix orpremix constituents.

Preferably the feed may be a fodder, or a premix thereof, a compoundfeed, or a premix thereof. In one embodiment the feed additivecomposition according to the present invention may be admixed with acompound feed, a compound feed component or to a premix of a compoundfeed or to a fodder, a fodder component, or a premix of a fodder.

The term “fodder” as used herein means any food which is provided to ananimal (rather than the animal having to forage for it themselves).Fodder encompasses plants that have been cut.

The term fodder includes silage, compressed and pelleted feeds, oils andmixed rations, and also sprouted grains and legumes.

Fodder may be obtained from one or more of the plants selected from:corn (maize), alfalfa (Lucerne), barley, birdsfoot trefoil, brassicas,Chau moellier, kale, rapeseed (canola), rutabaga (swede), turnip,clover, alsike clover, red clover, subterranean clover, white clover,fescue, brome, millet, oats, sorghum, soybeans, trees (pollard treeshoots for tree-hay), wheat, and legumes.

The term “compound feed” means a commercial feed in the form of a meal,a pellet, nuts, cake or a crumble. Compound feeds may be blended fromvarious raw materials and additives. These blends are formulatedaccording to the specific requirements of the target animal.

Compound feeds can be complete feeds that provide all the daily requirednutrients, concentrates that provide a part of the ration (protein,energy) or supplements that only provide additional micronutrients, suchas minerals and vitamins.

The main ingredients used in compound feed are the feed grains, whichinclude corn, wheat, canola meal, rapeseed meal, lupin, soybeans,sorghum, oats, and barley.

Suitably a premix as referred to herein may be a composition composed ofmicroingredients such as vitamins, minerals, chemical preservatives,antibiotics, fermentation products, and other essential ingredients.Premixes are usually compositions suitable for blending into commercialrations.

Any feedstuff of the present invention may comprise one or more feedmaterials selected from the group comprising a) cereals, such as smallgrains (e.g., wheat, barley, rye, oats, triticale and combinationsthereof) and/or large grains such as maize or sorghum; b) by productsfrom cereals, such as corn gluten meal, wet-cake (particularly cornbased wet-cake), Distillers Dried Grain (DDG) (particularly corn basedDistillers Dried Grain (cDDG)), Distillers Dried Grain Solubles (DDGS)(particularly corn based Distillers Dried Grain Solubles (cDDGS)), wheatbran, wheat middlings, wheat shorts, rice bran, rice hulls, oat hulls,palm kernel, and citrus pulp; c) protein obtained from sources such assoya, sunflower, peanut, lupin, peas, fava beans, cotton, canola, fishmeal, dried plasma protein, meat and bone meal, potato protein, whey,copra, sesame; d) oils and fats obtained from vegetable and animalsources; e) minerals and vitamins.

In one embodiment the feedstuff comprises or consists of corn, DDGS(such as cDDGS), wheat, wheat bran or a combination thereof.

In one embodiment the feed component may be corn, DDGS (e.g. cDDGS),wheat, wheat bran or a combination thereof.

In one embodiment the feedstuff comprises or consists of corn, DDGS(such as cDDGS) or a combination thereof.

In one embodiment a feed component may be corn, DDGS (such as cDDGS) ora combination thereof.

A feedstuff of the present invention may contain at least 30%, at least40%, at least 50% or at least 60% by weight corn and soybean meal orcorn and full fat soy, or wheat meal or sunflower meal.

A feedstuff of the present invention may contain between about 5 toabout 40% corn DDGS. For poultry—the feedstuff on average may containbetween about 7 to 15% corn DDGS. For swine (pigs)—the feedstuff maycontain on average 5 to 40% corn DDGS.

A feedstuff of the present invention may contain corn as a single grain,in which case the feedstuff may comprise between about 35% to about 80%corn.

In feedstuffs comprising mixed grains, e.g. comprising corn and wheatfor example, the feedstuff may comprise at least 10% corn.

In addition or in the alternative, a feedstuff of the present inventionmay comprise at least one high fibre feed material and/or at least oneby-product of the at least one high fibre feed material to provide ahigh fibre feedstuff. Examples of high fibre feed materials include:wheat, barley, rye, oats, by products from cereals, such as corn glutenmeal, corn gluten feed, wet-cake, Distillers Dried Grain (DDG),Distillers Dried Grain Solubles (DDGS), wheat bran, wheat middlings,wheat shorts, rice bran, rice hulls, oat hulls, palm kernel, and citruspulp. Some protein sources may also be regarded as high fibre: proteinobtained from sources such as sunflower, lupin, fava beans and cotton.

In one embodiment the feedstuff of the present invention comprises at,least one high fibre material and/or at least one by-product of the atleast one high fibre feed material selected from the group consisting ofDistillers Dried Grain Solubles (DDGS)—particularly cDDGS, wet-cake,Distillers Dried Grain (DDG)—particularly cDDG, wheat bran, and wheatfor example.

In one embodiment the feedstuff of the present invention comprises atleast one high fibre material and/or at least one by-product of the atleast one high fibre feed material selected from the group consisting ofDistillers Dried Grain Solubles (DDGS)—particularly cDDGS, wheat bran,and wheat for example.

In the present invention the feed may be one or more of the following: acompound feed and premix, including pellets, nuts or (cattle) cake; acrop or crop residue: corn, soybeans, sorghum, oats, barley copra,straw, chaff, sugar beet waste; fish meal; meat and bone meal; molasses;oil cake and press cake; oligosaccharides; conserved forage plants:silage; seaweed; seeds and grains, either whole or prepared by crushing,milling etc.; sprouted grains and legumes; yeast extract.

The term “feed” in the present invention encompasses in some embodimentspet food. A pet food is plant or animal material intended forconsumption by pets, such as dog food or cat food. Pet food, such as dogand cat food, may be either in a dry form, such as kibble for dogs, orwet canned form. Cat food may contain the amino acid taurine.

The term “feed” in the present invention encompasses in some embodimentsfish food. A fish food normally contains macro nutrients, trace elementsand vitamins necessary to keep captive fish in good health. Fish foodmay be in the form of a flake, pellet or tablet. Pelleted forms, some ofwhich sink rapidly, are often used for larger fish or bottom feedingspecies. Some fish foods also contain additives, such as beta caroteneor sex hormones, to artificially enhance the color of ornamental fish.

The term “feed” in the present invention encompasses in some embodimentbird food. Bird food includes food that is used both in birdfeeders andto feed pet birds. Typically bird food comprises of a variety of seeds,but may also encompass suet (beef or mutton fat).

As used herein the term “contacted” refers to the indirect or directapplication of the enzyme (or composition comprising the enzyme) of thepresent invention to the product (e.g. the feed). Examples of theapplication methods which may be used, include, but are not limited to,treating the product in a material comprising the feed additivecomposition, direct application by mixing the feed additive compositionwith the product, spraying the feed additive composition onto theproduct surface or dipping the product into a preparation of the feedadditive composition.

In one embodiment the feed additive composition of the present inventionis preferably admixed with the product (e.g. feedstuff). Alternatively,the feed additive composition may be included in the emulsion or rawingredients of a feedstuff.

For some applications, it is important that the composition is madeavailable on or to the surface of a product to be affected/treated. Thisallows the composition to impart one or more of the following favourablecharacteristics: performance benefits.

The enzyme (or composition comprising the enzyme) of the presentinvention may be applied to intersperse, coat and/or impregnate aproduct (e.g. feedstuff or raw ingredients of a feedstuff) with acontrolled amount of said enzyme.

Preferably, the enzyme (or composition comprising the enzyme) of thepresent invention will be thermally stable to heat treatment up to about70° C.; up to about 85° C.; or up to about 95° C. The heat treatment maybe performed for up to about 1 minute; up to about 5 minutes; up toabout 10 minutes; up to about 30 minutes; up to about 60 minutes. Theterm thermally stable means that at least about 75% of the enzyme thatwas present/active in the additive before heating to the specifiedtemperature is still present/active after it cools to room temperature.Preferably, at least about 80% of the enzyme that is present and activein the additive before heating to the specified temperature is stillpresent and active after it cools to room temperature.

In a particularly preferred embodiment the enzyme (or compositioncomprising the enzyme) of the present invention is homogenized toproduce a powder.

In an alternative preferred embodiment, the enzyme (or compositioncomprising the enzyme) of the present invention is formulated togranules as described in WO2007/044968 (referred to as TPT granules) orWO1997/016076 or WO1992/012645 incorporated herein by reference.

In another preferred embodiment when the feed additive composition isformulated into granules the granules comprise a hydrated barrier saltcoated over the protein core. The advantage of such salt coating isimproved thermo-tolerance, improved storage stability and protectionagainst other feed additives otherwise having adverse effect on theenzyme.

Preferably, the salt used for the salt coating has a water activitygreater than 0.25 or constant humidity greater than 60% at 20° C.

Preferably, the salt coating comprises a Na₂SO₄.

The method of preparing an enzyme (or composition comprising the enzyme)of the present invention may also comprise the further step of pelletingthe powder. The powder may be mixed with other components known in theart. The powder, or mixture comprising the powder, may be forced througha die and the resulting strands are cut into suitable pellets ofvariable length.

Optionally, the pelleting step may include a steam treatment, orconditioning stage, prior to formation of the pellets. The mixturecomprising the powder may be placed in a conditioner, e.g. a mixer withsteam injection. The mixture is heated in the conditioner up to aspecified temperature, such as from 60-100° C., typical temperatureswould be 70° C., 80° C., 85° C., 90° C. or 95° C. The residence time canbe variable from seconds to minutes and even hours. Such as 5 seconds,10 seconds, 15 seconds, 30 seconds, 1 minutes 2 minutes, 5 minutes, 10minutes, 15 minutes, 30 minutes and 1 hour.

It will be understood that the enzyme (or composition comprising theenzyme) of the present invention is suitable for addition to anyappropriate feed material.

It will be understood by the skilled person that different animalsrequire different feedstuffs, and even the same animal may requiredifferent feedstuffs, depending upon the purpose for which the animal isreared.

Optionally, the feedstuff may also contain additional minerals such as,for example, calcium and/or additional vitamins.

Preferably, the feedstuff is a corn soybean meal mix.

In one embodiment, preferably the feed is not pet food.

In another aspect there is provided a method for producing a feedstuff.Feedstuff is typically produced in feed mills in which raw materials arefirst ground to a suitable particle size and then mixed with appropriateadditives. The feedstuff may then be produced as a mash or pellets; thelater typically involves a method by which the temperature is raised toa target level and then the feed is passed through a die to producepellets of a particular size. The pellets are allowed to cool.Subsequently liquid additives such as fat and enzyme may be added.Production of feedstuff may also involve an additional step thatincludes extrusion or expansion prior to pelleting—in particular bysuitable techniques that may include at least the use of steam.

The feedstuff may be a feedstuff for a monogastric animal, such aspoultry (for example, broiler, layer, broiler breeders, turkey, duck,geese, water fowl), and swine (all age categories), a ruminant such ascattle (e.g. cows or bulls (including calves)), horses, sheep, a pet(for example dogs, cats) or fish (for example agastric fish, gastricfish, freshwater fish such as salmon, cod, trout and carp, e.g. koicarp, marine fish such as sea bass, and crustaceans such as shrimps,mussels and scallops). Preferably the feedstuff is for poultry.

Corn Based Feedstuff

In a preferred embodiment the feedstuff may be a corn based feedstuff.The term “corn based feedstuff” as used herein means a feedstuff whichcomprises or consists of corn (maize) or a by-product of corn.

Preferably the corn based feedstuff comprises corn or a by-product ofcorn as the major constituent. For example the corn based feedstuff maycomprise at least 35% corn or a by-product of corn, such as at least 40%corn or a by-product of corn, such as at least 50% corn or a by-productof corn, such as at least 60% corn or a by-product of corn, such as atleast 70% corn or a by-product of corn, such as at least 80% or aby-product of corn, such as at least 90% corn or a by-product of corn,for example 100% corn or a by-product of corn.

In some embodiments the corn based feedstuff may comprise corn or aby-product of corn as a minor constituent; in which case the feedstuffmay be supplemented with corn or a by-product of corn. By way of exampleonly the feedstuff may comprise for example wheat supplemented with cornor a by-product of corn.

When corn or the by-product of corn is a minor constituent of thefeedstuff, the corn or by-product of corn is at least 5%, preferably atleast 10%, preferably at least 20%, preferably at least 30% of thefeedstuff.

For the avoidance of doubt the term “corn” as used herein is synonymouswith maize, e.g. Zea mays.

In one embodiment the by-product of corn may be corn Distillers DriedGrain Solubles (cDDGS) or corn wet-cake or corn Distillers Dried Grain(DDG) or corn gluten meal or corn gluten feed or combinations thereof.

In one embodiment preferably the arabinoxylan-containing material of thepresent invention comprises a by-product of corn, such as cornDistillers Dried Grain Solubles (cDDGS) or corn wet-cake or cornDistillers Dried Grain (DDG) or corn gluten meal or corn gluten feed orcombinations thereof.

Wheat Based Feedstuff

In a preferred embodiment the feedstuff may be a wheat based feedstuff.The term “wheat based feedstuff” as used herein means a feedstuff whichcomprises or consists of wheat or a by-product of wheat.

Preferably the wheat based feedstuff comprises wheat or a by-product ofwheat as the major constituent. For example the wheat based feedstuffmay comprise at least 40% wheat or a by-product of wheat, such as atleast 60% wheat or a by-product of wheat, such as at least 80% or aby-product of wheat, such as at least 90% wheat or a by-product ofwheat, for example 100% wheat or a by-product of wheat.

In some embodiments the wheat based feedstuff may comprise wheat or aby-product of wheat as a minor constituent; in which case the feedstuffmay be supplemented with wheat or a by-product of wheat. By way ofexample only the feedstuff may comprise for example wheat supplementedwith wheat or a by-product of wheat.

When wheat or the by-product of wheat is a minor constituent of thefeedstuff, the wheat or by-product of wheat is at least 5%, preferablyat least 10%, preferably at least 20%, preferably at least 30% of thefeedstuff.

In one embodiment the by-product of wheat may be wheat bran, wheatmiddlings, wheat fibres for example.

Bran is the hard outer layer of grain and consists of combined aleuroneand pericarp. Along with germ, it is an integral part of whole grains,and is often produced as a by-product of milling in the production ofrefined grains. When bran is removed from grains, the grains lose aportion of their nutritional value. Bran is present in and may be milledfrom any cereal grain, including rice, corn (maize), wheat, oats, barleyand millet. Bran is particularly rich in dietary fiber and essentialfatty acids and contains significant quantities of starch, protein,vitamins and dietary minerals.

Wheat middlings is coarse and fine particles of wheat bran and fineparticles of wheat shorts, wheat germ, wheat flour and offal from the“tail of the mill”.

Wheat middlings is an inexpensive by-product intermediate of human foodand animal feed. In one embodiment preferably thearabinoxylan-containing material of the present invention compriseswheat bran and/or wheat middlings.

Wet-Cake, Distillers Dried Grains (DDG) and Distillers Dried GrainSolubles (DDGS)

Wet-cake, Distillers Dried Grains and Distillers Dried Grains withSolubles are products obtained after the removal of ethyl alcohol bydistillation from yeast fermentation of a grain or a grain mixture bymethods employed in the grain distilling industry.

Stillage coming from the distillation (e.g. comprising water, remainingsof the grain, yeast cells etc.) is separated into a “solid” part and aliquid part.

The solid part is called “wet-cake” and can be used as animal feed assuch.

The liquid part is (partially) evaporated into a syrup (solubles).

When the wet-cake is dried it is Distillers Dried Grains (DDG).

When the wet-cake is dried together with the syrup (solubles) it isDistillers Dried Grans with Solubles (DDGS).

Wet-cake may be used in dairy operations and beef cattle feedlots.

The dried DDGS may be used in livestock, e.g. dairy, beef and swine)feeds and poultry feeds.

Corn DDGS is a very good protein source for dairy cows.

Corn Gluten Meal

In one aspect, the by-product of corn may be corn gluten meal (CGM).

CGM is a powdery by-product of the corn milling industry. CGM hasutility in, for example, animal feed. It can be used as an inexpensiveprotein source for feed such as pet food, livestock feed and poultryfeed. It is an especially good source of the amino acid cysteine, butmust be balanced with other proteins for lysine.

Feed Additive Composition

The feed additive composition of the present invention and/or thefeedstuff comprising same may be used in any suitable form.

The feed additive composition of the present invention may be used inthe form of solid or liquid preparations or alternatives thereof.Examples of solid preparations include powders, pastes, boluses,capsules, pellets, tablets, dusts, and granules which may be wettable,spray-dried or freeze-dried. Examples of liquid preparations include,but are not limited to, aqueous, organic or aqueous-organic solutions,suspensions and emulsions.

In some applications, the feed additive compositions of the presentinvention may be mixed with feed or administered in the drinking water.

In one aspect the present invention relates to a method of preparing afeed additive composition, comprising admixing a xylanase as taughtherein with a feed acceptable carrier, diluent or excipient, and(optionally) packaging.

Premix

The feedstuff and/or feed additive composition may be combined with atleast one mineral and/or at least one vitamin. The compositions thusderived may be referred to herein as a premix.

Malting and Brewing

The enzyme (or composition comprising the enzyme) of the presentinvention may be used in malting and brewing.

Barley grains contain 1.7 to 4.1% (w/w) water-extractabie and 3.6 to6.4% (w/w) total beta-glucan (Anderson, M. A., Cook, J. A., & Stone, B.A., Journal of the Institute of Brewing, 1978, 84, 233-239; Henry, J.,Journal of the Science of Food and Agriculture, 1985, 36, 1243).

Wheat grains contain 0.1 to 0.8% (w/w) water-extractable and 0.6 to 1.4%(w/w) total beta-glucan (Anderson, M. A, et al (1978) supra).

Efficient hydrolysis of arabinoxylans (AXsol) and beta-glucan isimportant because such compounds can be involved in production problemssuch as wort viscosity (Ducroo, P. & Frelon, P. G., Proceedings of theEuropean Brewery Convention Congress, Zurich, 1989, 445; Viëtor, R. J. &Voragen, A. G. J., Journal of the Institute of Brewing, 1993, 99, 243)and filterability and haze formation (Coote, N. & Kirsop, B. H. 1976,Journal of the Institute of Brewing, 1976, 82, 34; Izawa, M., Kano, Y. &Kanimura, M. 1991. Proceedings Aviemore Conference on Malting, brewingand Distilling, 1990, 427).

The present invention provides a method of hydrolysing arabinoxylans(e.g. AXinsol and AXsol) during malting and brewing wherein wheatgrains, barley grains or a combination thereof, or portions of the wheatand/or barley grains, are admixed with the enzyme of the presentinvention.

In one aspect of the present invention may relate to a food compositionthat is a beverage, including, but not limited to, a fermented beveragesuch as beer and wine, comprising a xylanase comprising (or consistingof) a polypeptide sequence shown herein as SEQ ID No. 1, SEQ ID No. 2,SEQ ID No. 3, SEQ ID No. 9, SEQ ID No. 10, SEQ ID No. 11 or SEQ ID No.15, or a variant, homologue, fragment or derivative thereof having atleast 75% identity (such as at least 80%, 85%, 90%, 95%, 98% or 99%identity) with SEQ ID No. 1 or SEQ ID No. 2, SEQ ID No. 3, SEQ ID No. 9,SEQ ID No. 10, SEQ ID No. 11 or SEQ ID No. 15; or a polypeptide sequencewhich comprises SEQ ID No. 1, SEQ ID No. 2, SEQ ID No. 3, SEQ ID No. 9,SEQ ID No. 10, SEQ ID No. 11 or SEQ ID No. 15 with a conservativesubstitution of at least one of the amino acids; or a xylanase which isencoded by a nucleotide sequence shown herein as SEQ ID No. 4, SEQ IDNo. 5, SEQ ID No. 6, SEQ ID No. 12, SEQ ID No. 13, SEQ ID No. 14, SEQ IDNo. 16, SEQ ID No. 17 or SEQ ID No. 18, or a nucleotide sequence whichcan hybridize to SEQ ID No. 4, SEQ ID No. 5, SEQ ID No. 6, SEQ ID No.12, SEQ ID No. 13, SEQ ID No. 14, SEQ ID No. 16, SEQ ID No. 17 or SEQ IDNo. 18 under high stringency conditions, or a nucleotide sequence whichhas at least 75% identity (such as at least 80%, 85%, 90%, 95% or 98%identity) with SEQ ID No. 4, SEQ ID No. 5, SEQ ID No. 6, SEQ ID No. 12,SEQ ID No. 13, SEQ ID No. 14, SEQ ID No. 16, SEQ ID No. 17 or SEQ ID No.18; or a nucleotide sequence which differs from SEQ ID No. 4 or SEQ IDNo. 5 or SEQ ID No. 6 or SEQ ID No. 12 or SEQ ID No. 13 or SEQ ID No. 14or SEQ ID No. 16 or SEQ ID No. 17 or SEQ ID No. 18 due to the degeneracyof the genetic code.

In another aspect of the present invention may relate to a foodcomposition that is a beverage, including, but not limited to, afermented beverage such as beer and wine, comprising a xylanase enzymecomprising (or consisting of) a polypeptide sequence shown herein as SEQID No. 1, SEQ ID No. 2 or SEQ ID No. 3 or a variant, homologue, fragmentor derivative thereof having at least 98.5% (e.g. at least 98.8 or 99 or99.1 or 99.5%) identity with SEQ ID No. 1 or SEQ ID No. 2 or SEQ ID No.3 or a xylanase which is encoded by a nucleotide sequence shown hereinas SEQ ID No. 4, SEQ ID No. 5 or SEQ ID No. 6, or a nucleotide sequencewhich has at least 97.7% (e.g. at least 98%, 98.5% or 99%) identity withSEQ ID No. 4, SEQ ID No. 5 or SEQ ID No. 6.

In the context of the present invention, the term “fermented beverage”is meant to comprise any beverage produced by a method comprising afermentation process, such as a microbial fermentation, such as abacterial and/or yeast fermentation.

In an aspect of the invention the fermented beverage is beer. The term“beer” is meant to comprise any fermented wort produced byfermentation/brewing of a starch-containing plant material. Often, beeris produced from malt or adjunct, or any combination of malt and adjunctas the starch-containing plant material. As used herein the term “malt”is understood as any malted cereal grain, such as malted barley orwheat.

As used herein the term “adjunct” refers to any starch and/or sugarcontaining plant material which is not malt, such as barley or wheatmalt. As examples of adjuncts, mention can be made of materials such ascommon corn grits, refined corn grits, brewer's milled yeast, rice,sorghum, refined corn starch, barley, barley starch, dehusked barley,wheat, wheat starch, fortified cereal, cereal flakes, rye, oats, corn(maize), potato, tapioca, cassava and syrups, such as corn syrup, sugarcane syrup, inverted sugar syrup, barley and/or wheat syrups, and thelike may be used as a source of starch.

As used herein, the term “mash” refers to an aqueous slurry of anystarch and/or sugar containing plant material such as grist, e. g.comprising crushed barley malt, crushed barley, and/or other adjunct ora combination hereof, mixed with water later to be separated into wortand spent grains.

As used herein, the term “wort” refers to the unfermented liquor run-offfollowing extracting the grist during mashing.

In another aspect the invention relates to a method of preparing afermented beverage such as beer comprising mixing the xylanase of thepresent invention with malt or adjunct.

Examples of beers comprise: full malted beer, beer brewed under the“Reinheitsgebot”, ale, IPA, lager, bitter, Happoshu (second beer), thirdbeer, dry beer, near beer, light beer, low alcohol beer, low caloriebeer, porter, bock beer, stout, malt liquor, non-alcoholic beer,non-alcoholic malt liquor and the like, but also alternative cereal andmalt beverages such as fruit flavoured malt beverages, e. g. citrusflavoured, such as lemon-, orange-, lime-, or berry-flavoured maltbeverages, liquor flavoured malt beverages, e. g., vodka-, rum-, ortequila-flavoured malt liquor, or coffee flavoured malt beverages, suchas caffeine-flavoured malt liquor, and the like.

Breakdown of Grain-Based Material e.g. For Biofuel Production

The enzyme (or composition comprising the enzyme) of the presentinvention or as disclosed herein may be used to breakdown (degrade)AXinsol and AXsol during grain processing from e.g. grain-basedmaterial. The grain-based material may be whole grains (e.g. wholewheat, barley, rye, triticale or corn grains or mixtures thereof) orportions of the whole grains, or mixtures thereof.

In one embodiment the enzyme (or composition comprising the enzyme) ofthe present invention or as disclosed herein may be used to breakdown(degrade) AXinsol and AXsol in grain-based materials or whole grains.

For the avoidance of doubt the whole grains can be mechanically broken.

The grain-based material may be broken down or degraded to glucose. Theglucose may subsequently be used as a feedstock for any fermentationprocess, e.g. for biofuel (e.g. bioethanol) production and/orbiochemicals (e.g., bio-based isoprene) production.

The grain-based material may be feedstock for a biofuel (e.g.bioethanol) production process.

Today most fuel ethanol is produced from corn (maize) grain, which ismilled, treated with amylase enzymes to hydrolyse starch to sugars,fermented, and distilled. While substantial progress has been made inreducing costs of ethanol production, substantial challenges remain.Improved techniques are still needed to reduce the cost of biofuelfeedstocks for ethanol production. For example, in grain-based ethanolproduction degradation of arabinoxylans may increase accessibility ofstarch.

The present invention provides a xylanase for use in the breakdown ofhemicelluloses, e.g. arabinoxylan—particularly AXinsol and AXsol.

By way of example only, in the European fuel alcohol industry, smallgrains like wheat, barley and rye are common raw materials, in the UScorn is mainly used. Wheat, barley and rye contain, next to starch, highlevels of non-starch polysaccharide polymers (NSP), like cellulose,beta-glucan and hemicellulose.

The ratio in which the different NSPs are represented differ for eachfeedstock. The table below shows the different amounts of NSPs in wheat,barley and rye compared to some other feedstocks.

TABLE 1 Non-starch Polysaccharides present in different feedstocks (gkg⁻¹ dry matter) Barley Oats Corn Wheat Rye Hulled Hulless HulledHulless Beta- 1 8 16 42 42 28 41 Glucan Cellulose 22 17-20 15-16 43 1082 14 Soluble 75 89-99 116-136 144 114 150 113 and Non- soluble NCP¹Total NSP 97 107-119 132-152 186 124 232 116 ¹Non CellulosicPolysaccharides: pentosans, (arabino)xylans and other hemicelluloses

NSPs can give high viscosity to grain mashes due to their largewater-binding capacity. High viscosity has a negative impact on ethanolproduction since it will limit the solid concentration that can be usedin mashing and it will reduce the energy efficiency of the process. Inaddition, residual hemicelluloses present throughout the process maycontribute to fouling in heat exchangers and distillation equipment. Thelargest impact of a high viscosity is seen when a mash is cooled tofermentation temperature (32° C.). This explains that the viscosityneeds to be reduced in the process anywhere before the cooling step.

In one embodiment of the present invention the method for degradinggrain-based material comprises admixing the xylanase as disclosed hereinas early as possible in the biofuel (e.g. bioethanol) productionprocess, e.g. preferably during mixing of the grain-based material atthe start of the process. One advantage of adding the xylanases asdisclosed herein at an early stage in the process is that the enzymesbreakdown initial viscosity.

In one embodiment of the present invention the method for degradinggrain-based material comprises admixing the xylanase as disclosed hereinprior to or during saccharification, fermentation, or a combinationthereof.

In one embodiment of the present invention the method for degradinggrain-based material comprises admixing the xylanase as disclosed hereinduring liuquefaction (e.g. a high temperature step that follows mixing).

Therefore in one embodiment the present invention relates to reducingviscosity when degrading grain-based materials, e.g. in biofuel (e.g.bioethanol) production processes.

The benefits of using the xylanases taught herein to reduce viscositywhen degrading grain-based materials, e.g. in biofuel (e.g. bioethanol)production processes are multiple:

-   -   Higher dry substance mash can be used in the process    -   Higher solids content of final syrup can be obtained    -   Better heat transfer, lower energy requirement    -   Reduced evaporator fouling leading to reduced cleaning costs    -   Increased final ethanol yields    -   Improved quality of DDGS (by-product)    -   Better separation between the solid and liquid part during        stillage separation (after distillation). The lower viscosity        increases separation efficiency.

A further significant advantage of the present invention is that use ofthe xylanase described herein in biofuel production can also result inimproved (by)products from that process such as wet-cake, DistillersDried Grains (DDG) or Distillers Dried Grains with Solubles (DDGS).Therefore one advantage of the present invention is since the wet-cake,DDG and DDGS are (by)products of biofuel (e.g. bioethanol) productionthe use of the present invention can result is improved quality of these(by)products. For example the arabinoxylans in the (by)products can bealready dissolved during the biofuel production process.

Cereal (e.g. Wheat) Gluten-Starch Separation

The enzyme (or composition comprising the enzyme) of the presentinvention or as disclosed herein may be used to breakdown (degrade)AXinsol and AXsol during wheat starch and gluten separation.

After initial separation of the wheat bran and germ from the endosperm,fractionation of wheat endosperm flour into starch and gluten fractionsis industrially applied on large scale to obtain high quality A-starchand byproducts B-starch and vital gluten.

The product of the degradation of the cereal flour (e.g. wheat flour) inthe present invention is starch (high quality A-starch).

In addition, by-products B-starch and vital gluten are also produced.Each individual product is then further processed to supplement ormodify food product characteristics to the market needs.

There are several wheat separation processes used by industry describedin literature. These industrial processes differ mainly in the forms ofthe flour-water mixtures presented to the fractionation equipment(centrifuge, hydrocyclone, or screen) or in the initial reactionconditions as temperature and applying of shear (Abdulvahit Sayaslan,Lebensm-Wiss. U.—Technol 37 (2004) 499-515, Wetmilling of wheat flourindustrial processes and small-scale test methods).

In the method for separating a cereal flour (e.g. wheat flour) intostarch and gluten fractions the method comprises admixing a cereal flour(e.g. wheat flour), water and a xylanase. The cereal flour, water andxylanase may be mixed simultaneously or sequentially. In someembodiments the cereal flour (e.g. wheat flour) and water may be admixedbefore admixing with the xylanase.

In general, cereal flour (e.g. wheat flour) is either mixed to a doughor batter, varying between 35 to 63% Dry solids, at temperatures of˜20-45° C. The mixture is then further processed either by:

-   -   1) letting the mixture rest for some time (˜30 minutes) and        sequentially washing out the starch from the mixture using a        screen, centrifuge or hydrocyclone to separate the starch milk        from the gluten, or    -   2) applying shear to the mixture, optionally diluting the        mixture further and then separating the wheat flour by a        hydrocyclone, or a 2- or 3-phase decanter centrifuge.

The term “dry solids” as used herein means total solids (dissolved andundissolved) of a slurry (in %) on a dry weight basis.

In one embodiment of the present invention the method or use as claimedmay include the steps of mixing wheat flour to form a dough or batterbetween 35-63% dry solids, at a temperature of about 20 to about 45° C.and separating the starch from the gluten.

The method of the present invention may further comprise:

-   a) resting the mixture for about 30 minutes and sequentially washing    out the starch from the mixture using either a screen, a centrifuge    or a hydrocyclone to separate the starch milk from the gluten; or-   b) applying shear to the mixture and optionally diluting the mixture    further, separating the starch from the gluten using a hydrocyclone    or a 2- or 3-phase decanter centrifuge.

The present invention provides for improving the separation of thestarch and the gluten by adding xylanases as taught herein suitablyduring the initial mixing step of flour and water in the variousprocesses described above used for wheat starch separation. Separationis improved by adding xylanases during the initial mixing step due toviscosity reduction and the hydrolysis of AXsol and/or AXinsolinterfering with the gluten particles. By degrading these poly- andoligosaccharides, gluten agglomeration is enhanced, improving the glutenyield. (S. A. Frederix, C. M. Courtin, J. A. Delcour, J. Cereal Sci. 40(2004) 41-49, Substrate selectivity and inhibitor sensitivity affectxylanase functionality in wheat flour gluten-starch separation).

One advantage of the present invention is that it results in higherA-starch yields and/or better quality gluten (e.g. better quality vitalgluten).

One advantage of the present invention is that it improves wheatgluten-starch separation.

One of the ways to evaluate gluten quality is by monitoring glutenagglomeration. When a certain amount of friction through kneading of thedough or mixing of the batter is applied, gluten particles tend toagglomerate into larger particles that form a polymeric network, called“vital gluten”. “Vital gluten” can be added to food products to improveproperties of baked goods such as dough strength, shelf-life and breadvolume (L. Day, M. A. Augustin, I. L. Batey and C. W. Wrigley;Wheat-gluten uses and industry needs; Trends in Food Science &Technology 17 (2006) 82-90).

In the bakery industry, the quality and quantity of the gluten in awheat flour is determined by the ICC standard assay No. 155 (AACC 38-12)using a Glutomatic. In this device, a dough is formed from wheat flour(10.0 gr) mixed with a small amount of 2% NaCl solution (4.2-4.8 ml).After 20 seconds of mixing step, the dough is continuously kneaded whilebeing washed for 5 minutes with a 2% NaCl solution at room temperature(˜22° C.) pumped through the mixing cup at a flow rate of ˜70 ml/minute.During this washing step, the wash water containing starch is collectedand the gluten particles form a gluten ball within the Glutomatic sieveholder.

The quality of the gluten is measured by evaluating the glutenagglomeration. This is done by centrifuging the gluten ball in a specialcentrifuge containing a small sieve. The gluten particles that pass thissieve are weighed (small gluten) and the total amount of gluten isweighed. The gluten index is calculated by (total wet gluten−small wetgluten)/total wet gluten. The more gluten agglomeration is improved, thesmaller the small gluten fraction will be and the higher the glutenindex value is. A high gluten index, with a theoretical maximum of 100%,indicates a high quality gluten ball.

Another value to quantify the amount of gluten is the dried gluten yield(%). This value is calculated by dividing the grams of total driedgluten by the total amount of dry flour which was used in theexperiment. The more dried gluten is recovered, the better theseparation is. This industrial assay is currently under adaptation tosimulate a dough separation process used in industry.

Dosages

Preferably, the xylanase is present in the xylan-containing material(e.g. feedstuff) in the range of about 500 XU/kg to about 16,000 XU/kgxylan-containing material (e.g. feed), more preferably about 750 XU/kgfeed to about 8000 XU/kg xylan-containing material (e.g. feed),preferably about 1500 XU/kg feed to about 3000 XU/kg xylan-containingmaterial (e.g. feed), preferably about 2000 XU/kg feed to about 2500XU/kg xylan-containing material (e.g. feed), and even more preferablyabout 1000 XU/kg xylan-containing material (e.g. feed) to about 4000XU/kg xylan-containing material (e.g. feed).

In one embodiment the xylanase is present in the xylan-containingmaterial (e.g. feedstuff) at more than about 500 XU/kg xylan-containingmaterial (e.g. feed), suitably more than about 600 XU/kgxylan-containing material (e.g. feed), suitably more than about 700XU/kg xylan-containing material (e.g. feed), suitably more than about800 XU/kg xylan-containing material (e.g. feed), suitably more thanabout 900 XU/kg xylan-containing material (e.g. feed), suitably morethan about 1000 XU/kg xylan-containing material (e.g. feed), suitablymore than about 2000 XU/kg, suitably more than about 2500 XU/kg,suitably more than about 3000 XU/kg xylan-containing material (e.g.feed).

In one embodiment the xylanase is present in the xylan-containingmaterial (e.g. feedstuff) at a concentration of between about 2000 XU/kgto about 2500 XU/kg.

In one embodiment the xylanase is present in the xylan-containingmaterial (e.g. feedstuff) at less than about 16,000 XU/kgxylan-containing material (e.g. feed), suitably less than about 8000XU/kg xylan-containing material (e.g. feed), suitably less than about7000 XU/kg xylan-containing material (e.g. feed), suitably less thanabout 6000 XU/kg xylan-containing material (e.g. feed), suitably lessthan about 5000 XU/kg xylan-containing material (e.g. feed), suitablyless than about 4000 XU/kg xylan-containing material (e.g. feed).

Preferably, the xylanase may be present in a feed additive compositionin range of about 100 XU/g to about 320,000 XU/g composition, morepreferably about 300 XU/g composition to about 160,000 XU/g composition,and even more preferably about 500 XU/g composition to about 50,000 XU/gcomposition, and even more preferably about 500 XU/g composition toabout 40,000 XU/g composition.

In one embodiment the xylanase is present in the feed additivecomposition at more than about 100 XU/g composition, suitably more thanabout 200 XU/g composition, suitably more than about 300 XU/gcomposition, suitably more than about 400 XU/g composition, suitablymore than about 500 XU/g composition.

In one embodiment the xylanase is present in the feed additivecomposition at less than about 320,000 XU/g composition, suitably lessthan about 160,000 XU/g composition, suitably less than about 50,000XU/g composition, suitably less than about 40,000 XU/g composition,suitably less than about 30000 XU/g composition.

The xylanase activity can be expressed in xylanase units (XU) measuredat pH 5.0 with AZCL-arabinoxylan (azurine-crosslinked wheatarabinoxylan, Xylazyme tablets, Megazyme) as substrate. Hydrolysis byendo-(1-4)-β-D-xylanase (xylanase) produces water soluble dyedfragments, and the rate of release of these (increase in absorbance at590 nm) can be related directly to enzyme activity. The xylanase units(XU) are determined relatively to an enzyme standard (Danisco Xylanase,available from Danisco Animal Nutrition) at standard reactionconditions, which are 40° C., 5 min reaction time in McIlvaine buffer,pH 5.0.

The xylanase activity of the standard enzyme is determined as amount ofreleased reducing sugar end groups from an oat-spelt-xylan substrate permin at pH 5.3 and 50° C. The reducing sugar end groups react with 3,5-Dinitrosalicylic acid and formation of the reaction product can bemeasured as increase in absorbance at 540 nm. The enzyme activity isquantified relative to a xylose standard curve (reducing sugarequivalents). One xylanase unit (XU) is the amount of standard enzymethat releases 0.5 μmol of reducing sugar equivalents per min at pH 5.3and 50° C.

In one embodiment suitably the enzyme is classified using the E.C.classification above, and the E.C. classification designates an enzymehaving that activity when tested in the assay taught herein fordetermining 1 XU.

Preferably, the xylanase is present in the mixing step of a wheat starchseparation process in the dough or batter in the range of about 0.01kg/MT DS dough or batter to about 0.60 kg/MT DS, more preferably about0.05 kg/MT DS to about 0.45 kg/MT DS dough or batter, and even morepreferably about 0.10 kg/MT DS to about 0.25 kg/MT DS dough or batter.

In some embodiments (particularly in the wheat starch separationembodiment) the xylanase may be dosed in the range of about 0.019 gprotein/MT DS wheat flour (which is equivalent to 0.019 mg/kg DS) toabout 119 g protein/MT DS wheat flour (which is equivalent to 119 mg/kgDS—where DS means dry solids content and MT means metric ton.

In some embodiments (particularly in the wheat starch separationembodiment) the xylanase maybe dosed at about 1.19 g protein/MT DS wheatflour (which is equivalent to about 1.19 mg/kg DS)—where DS means drysolids content and MT means metric ton.

In some embodiments (particularly in the wheat starch separationembodiment) the xylanase may be dosed in the range of about 9 to about120000 units/kg wheat flour, suitably between about 500-2400 units/kgwheat flour, suitably between about 900-1200 units/kg wheat flour(wherein 1 unit is defined as the amount of enzyme required to generate1 micromole of xoylose reducing sugar equivalents per minute under theconditions of the birch wood assay of Example 3).

In some embodiments (particularly in degrading grain-based material) thexylanase may be dosed in the range of about 0.29 g/protein/MT DS wheat(which is equivalent to 0.29 mg/kg DS) to about 0290 g/protein/MT DSwheat (which is equivalent to 290 mg/kg DS).

In some embodiments (particularly in degrading grain-based material) thexylanase may be dosed at 2.9 g/protein/MT DS wheat (which is equivalentto 2.9 mg/kg DS).

In some embodiments (particularly in degrading grain-based material) thexylanase may be dosed in the range of about 22 to about 285000 units/kg,suitably about 1100 to about 5700 units/kg, suitably about 2200 to about2850 units/kg (wherein 1 unit is defined as the amount of enzymerequired to generate 1 micromole of xoylose reducing sugar equivalentsper minute under the conditions of the birch wood assay of Example 3).

The enzyme and/or composition comprising the enzyme according to thepresent invention may be designed for one-time dosing or may be designedfor use (e.g. feeding) on a daily basis.

The optimum amount of the enzyme and/or composition comprising theenzyme to be used in the present invention will depend on the product tobe treated and/or the method of contacting the product with thecomposition and/or the intended use for the same.

The amount of enzyme used in the compositions should be a sufficientamount to be effective.

The amount of enzyme used in the compositions should be a sufficientamount to be effective and to remain sufficiently effective in forexample improving the performance of an animal fed feed productscontaining said composition. This length of time for effectivenessshould extend up to at least the time of utilisation of the product(e.g. feed additive composition or feed containing same).

Formulation

In one embodiment the enzyme may be formulated as a liquid, a dry powderor a granule.

The dry powder or granules may be prepared by means known to thoseskilled in the art, such as, in top-spray fluid bed coater, in a buttonspray Wurster or by drum granulation (e.g. High sheer granulation),extrusion, pan coating or in a microingredients mixer.

For some embodiments the enzyme may be coated, for example encapsulated.

In one embodiment the coating protects the enzyme from heat and may beconsidered a thermoprotectant.

In one embodiment the feed additive composition is formulated to a drypowder or granules as described in WO2007/044968 (referred to as TPTgranules) or WO1997/016076 or WO1992/012645 (each of which isincorporated herein by reference).

In one embodiment the feed additive composition may be formulated to agranule for feed compositions comprising: a core; an active agent; andat least one coating, the active agent of the granule retaining at least50% activity, at least 60% activity, at least 70% activity, at least 80%activity after conditions selected from one or more of a) a feedpelleting process, b) a steam-heated feed pretreatment process, c)storage; d) storage as an ingredient in an unpelleted mixture, and e)storage as an ingredient in a feed base mix or a feed premix comprisingat least one compound selected from trace minerals, organic acids,reducing sugars, vitamins, choline chloride, and compounds which resultin an acidic or a basic feed base mix or feed premix.

With regard to the granule at least one coating may comprise a moisturehydrating material that constitutes at least 55% w/w of the granule;and/or at least one coating may comprise two coatings. The two coatingsmay be a moisture hydrating coating and a moisture barrier coating. Insome embodiments, the moisture hydrating coating may be between 25% and60% w/w of the granule and the moisture barrier coating may be between2% and 15% w/w of the granule. The moisture hydrating coating may beselected from inorganic salts, sucrose, starch, and maltodextrin and themoisture barrier coating may be selected from polymers, gums, whey andstarch.

The granule may be produced using a feed pelleting process and the feedpretreatment process may be conducted between 70° C. and 95° C. for upto several minutes, such as between 85° C. and 95° C.

In one embodiment the feed additive composition may be formulated to agranule for animal feed comprising: a core; an active agent, the activeagent of the granule retaining at least 80% activity after storage andafter a steam-heated pelleting process where the granule is aningredient; a moisture barrier coating; and a moisture hydrating coatingthat is at least 25% w/w of the granule, the granule having a wateractivity of less than 0.5 prior to the steam-heated pelleting process.

The granule may have a moisture barrier coating selected from polymersand gums and the moisture hydrating material may be an inorganic salt.The moisture hydrating coating may be between 25% and 45% w/w of thegranule and the moisture barrier coating may be between 2% and 10% w/wof the granule.

The granule may be produced using a steam-heated pelleting process whichmay be conducted between 85° C. and 95° C. for up to several minutes.

In some embodiments the enzyme may be diluted using a diluent, such asstarch powder, lime stone or the like.

In one embodiment, the enzyme or composition comprising the enzyme is ina liquid formulation suitable for consumption preferably such liquidconsumption contains one or more of the following: a buffer, salt,sorbitol and/or glycerol.

In another embodiment the enzyme or composition comprising the enzymemay be formulated by applying, e.g. spraying, the enzyme(s) onto acarrier substrate, such as ground wheat for example.

In one embodiment the enzyme or composition comprising the enzymeaccording to the present invention may be formulated as a premix. By wayof example only the premix may comprise one or more feed components,such as one or more minerals and/or one or more vitamins.

In one embodiment the enzyme for use in the present invention areformulated with at least one physiologically acceptable carrier selectedfrom at least one of maltodextrin, limestone (calcium carbonate),cyclodextrin, wheat or a wheat component, sucrose, starch, Na₂SO₄, Talc,PVA, sorbitol, benzoate, sorbiate, glycerol, sucrose, propylene glycol,1,3-propane diol, glucose, parabens, sodium chloride, citrate, acetate,phosphate, calcium, metabisulfite, formate and mixtures thereof.

Packaging

In one embodiment the enzyme and/or composition comprising same (e.g.feed additive composition) and/or premix and/or feed or feedstuffaccording to the present invention is packaged.

In one preferred embodiment the feed additive composition and/or premixand/or feed or feedstuff is packaged in a bag, such as a paper bag.

In an alternative embodiment the feed additive composition and/or premixand/or feed or feedstuff may be sealed in a container. Any suitablecontainer may be used.

Forms

The enzyme or composition comprising the enzyme (e.g. the feed additivecomposition) of the present invention and other components and/or thefeedstuff comprising same may be used in any suitable form.

The enzyme or composition comprising same (e.g. feed additivecomposition) of the present invention may be used in the form of solidor liquid preparations or alternatives thereof. Examples of solidpreparations include powders, pastes, boluses, capsules, pellets,tablets, pills, capsules, ovules, solutions or suspensions, dusts, andgranules which may be wettable, spray-dried or freeze-dried. Examples ofliquid preparations include, but are not limited to, aqueous, organic oraqueous-organic solutions, suspensions and emulsions.

The composition comprising the enzyme may contain flavouring orcolouring agents, for immediate-, delayed-, modified-, sustained-,pulsed- or controlled-release applications.

By way of example, if the composition of the present invention is usedin a solid, e.g. pelleted form, it may also contain one or more of:excipients such as microcrystalline cellulose, lactose, sodium citrate,calcium carbonate, dibasic calcium phosphate and glycine; disintegrantssuch as starch (preferably corn, potato or tapioca starch), sodiumstarch glycollate, croscarmellose sodium and certain complex silicates;granulation binders such as polyvinylpyrrolidone,hydroxypropylmethylcellulose (HPMC), hydroxypropylcellulose (HPC),sucrose, gelatin and acacia; lubricating agents such as magnesiumstearate, stearic acid, glyceryl behenate and talc may be included.

Examples of nutritionally acceptable carriers for use in preparing theforms include, for example, water, salt solutions, alcohol, silicone,waxes, petroleum jelly, vegetable oils, polyethylene glycols, propyleneglycol, liposomes, sugars, gelatin, lactose, amylose, magnesiumstearate, talc, surfactants, silicic acid, viscous paraffin, perfumeoil, fatty acid monoglycerides and diglycerides, petroethral fatty acidesters, hydroxymethyl-cellulose, polyvinylpyrrolidone, and the like.

Preferred excipients for the forms include lactose, starch, a cellulose,milk sugar or high molecular weight polyethylene glycols.

For aqueous suspensions and/or elixirs, the composition of the presentinvention may be combined with various sweetening or flavouring agents,colouring matter or dyes, with emulsifying and/or suspending agents andwith diluents such as water, propylene glycol and glycerin, andcombinations thereof.

Subject

The term “subject”, as used herein, means an animal that is to be or hasbeen administered with a feed additive composition according to thepresent invention or a feedstuff comprising said feed additivecomposition according to the present invention.

The term “subject”, as used herein, means an animal.

In one embodiment, the subject is a mammal, bird, fish or crustaceanincluding for example livestock or a domesticated animal (e.g. a pet).

In one embodiment the “subject” is livestock.

The term “livestock”, as used herein refers to any farmed animal.Preferably, livestock is one or more of ruminants such as cattle (e.g.cows or bulls (including calves)), mono-gastric animals such as poultry(including broilers, chickens and turkeys), pigs (including piglets),birds, aquatic animals such as fish, agastric fish, gastric fish,freshwater fish such as salmon, cod, trout and carp, e.g. koi carp,marine fish such as sea bass, and crustaceans such as shrimps, musselsand scallops), horses (including race horses), sheep (including lambs).

In another embodiment the “subject” is a domesticated animal or pet oran animal maintained in a zoological environment.

The term “domesticated animal or pet or animal maintained in azoological environment” as used herein refers to any relevant animalincluding canines (e.g. dogs), felines (e.g. cats); rodents (e.g. guineapigs, rats, mice), birds, fish (including freshwater fish and marinefish), and horses.

Performance

As used herein, “animal performance” may be determined by the feedefficiency and/or weight gain of the animal and/or by the feedconversion ratio and/or by the digestibility of a nutrient in a feed(e.g. amino acid digestibility) and/or digestible energy ormetabolizable energy in a feed and/or by nitrogen retention and/or byanimals ability to avoid the negative effects of necrotic enteritisand/or by the immune response of the subject.

Preferably “animal performance” is determined by feed efficiency and/orweight gain of the animal and/or by the feed conversion ratio.

By “improved animal performance” it is meant that there is increasedfeed efficiency, and/or increased weight gain and/or reduced feedconversion ratio and/or improved digestibility of nutrients or energy ina feed and/or by improved nitrogen retention and/or by an improvedimmune response in the subject resulting from the use of feed additivecomposition of the present invention in feed in comparison to feed whichdoes not comprise said feed additive composition.

Preferably, by “improved animal performance” it is meant that there isincreased feed efficiency and/or increased weight gain and/or reducedfeed conversion ratio.

As used herein, the term “feed efficiency” refers to the amount ofweight gain per unit of feed when the animal is fed ad-libitum or aspecified amount of feed during a period of time.

By “increased feed efficiency” it is meant that the use of a feedadditive composition according the present invention in feed results inan increased weight gain per unit of feed intake compared with an animalfed without said feed additive composition being present.

Feed Conversion Ratio (FCR)

As used herein, the term “feed conversion ratio” refers to the amount offeed fed to an animal to increase the weight of the animal by aspecified amount.

An improved feed conversion ratio means a lower feed conversion ratio.

By “lower feed conversion ratio” or “improved feed conversion ratio” itis meant that the use of a feed additive composition in feed results ina lower amount of feed being required to be fed to an animal to increasethe weight of the animal by a specified amount compared to the amount offeed required to increase the weight of the animal by the same amountwhen the feed does not comprise said feed additive composition.

Nutrient Digestibility

Nutrient digestibility as used herein means the fraction of a nutrientthat disappears from the gastro-intestinal tract or a specified segmentof the gastro-intestinal tract, e.g. the small intestine. Nutrientdigestibility may be measured as the difference between what isadministered to the subject and what comes out in the faeces of thesubject, or between what is administered to the subject and what remainsin the digesta on a specified segment of the gastro intestinal tract,e.g. the ileum,

Nutrient digestibility as used herein may be measured by the differencebetween the intake of a nutrient and the excreted nutrient by means ofthe total collection of excreta during a period of time; or with the useof an inert marker that is not absorbed by the animal, and allows theresearcher calculating the amount of nutrient that disappeared in theentire gastro-intestinal tract or a segment of the gastro-intestinaltract. Such an inert marker may be titanium dioxide, chromic oxide oracid insoluble ash. Digestibility may be expressed as a percentage ofthe nutrient in the feed, or as mass units of digestible nutrient permass units of nutrient in the feed.

Nutrient digestibility as used herein encompasses starch digestibility,fat digestibility, protein digestibility, and amino acid digestibility.

Energy digestibility as used herein means the gross energy of the feedconsumed minus the gross energy of the faeces or the gross energy of thefeed consumed minus the gross energy of the remaining digesta on aspecified segment of the gastro-intestinal tract of the animal, e.g. theileum. Metabolizable energy as used herein refers to apparentmetabolizable energy and means the gross energy of the feed consumedminus the gross energy contained in the faeces, urine, and gaseousproducts of digestion. Energy digestibility and metabolizable energy maybe measured as the difference between the intake of gross energy and thegross energy excreted in the faeces or the digesta present in specifiedsegment of the gastro-intestinal tract using the same methods to measurethe digestibility of nutrients, with appropriate corrections fornitrogen excretion to calculate metabolizable energy of feed.

Combination with Other Components

The enzyme of the present invention (or the xylanase as taught herein)may be used in combination with other components.

In one embodiment the enzyme of the present invention (or the xylanaseas taught herein) may be used in combination with a probiotic or adirect fed microbial (DFM), e.g. a direct fed bacteria.

The combination of the present invention comprises the enzyme of thepresent invention (or the xylanase as taught herein or a compositioncomprising the enzyme, e.g. a feed additive composition) and anothercomponent which is suitable for human or animal consumption and iscapable of providing a medical or physiological benefit to the consumer.

In one embodiment the “another component” may be one or more furtherenzymes (e.g. further feed enzymes or brewing or malting enzymes, orgrain processing enzymes or wheat gluten-starch separation enzymes).

Suitable additional enzymes for use in the present invention may be oneor more of the enzymes selected from the group consisting of:endoglucanases (E.C. 3.2.1.4); celliobiohydrolases (E.C. 3.2.1.91),β-glucosidases (E.C. 3.2.1.21), cellulases (E.C. 3.2.1.74), lichenases(E.C. 3.1.1.73), lipases (E.C. 3.1.1.3), lipid acyltransferases(generally classified as E.C. 2.3.1.x), phospholipases (E.C. 3.1.1.4,E.C. 3.1.1.32 or E.C. 3.1.1.5), phytases (e.g. 6-phytase (E.C. 3.1.3.26)or a 3-phytase (E.C. 3.1.3.8), alpha-amylases (E.C. 3.2.1.1), otherxylanases (E.C. 3.2.1.8, E.C. 3.2.1.32, E.C. 3.2.1.37, E.C. 3.1.1.72,E.C. 3.1.1.73), glucoamylases (E.G. 3.2.1.3), proteases (e.g. subtilisin(E.C. 3.4.21.62) or a bacillolysin (E.C. 3.4.24.28) or an alkalineserine protease (E.C. 3.4.21.x) or a keratinase (E.C. 3.4.x.x)) and/ormannanases (e.g. a β-mannanase (E.C. 3.2.1.78)).

In one embodiment (particularly for feed applications) the othercomponent may be one or more of the enzymes selected from the groupconsisting of an amylase (including α-amylases (E.C. 3.2.1.1),G4-forming amylases (E.C. 3.2.1.60), β-amylases (E.C. 3.2.1.2) andγ-amylases (E.C. 3.2.1.3); and/or a protease (e.g. subtilisin (E.C.3.4.21.62) or a bacillolysin (E.C. 3.4.24.28) or an alkaline serineprotease (E.C. 3.4.21.x) or a keratinase (E.C. 3.4.x.x)).

In one embodiment (particularly for feed applications) the othercomponent may be a combination of an amylase (e.g. α-amylases (E.C.3.2.1.1)) and a protease (e.g. subtilisin (E.C. 3.4.21.62)).

In one embodiment (particularly for feed applications) the othercomponent may be a β-glucanase, e.g. an endo-1,3(4)-β-glucanases (E.C.3.2.1.6).

In one embodiment (particularly for feed applications) the othercomponent may be a mannanases (e.g. a β-mannanase (E.C. 3.2.1.78)).

In one embodiment (particularly for feed applications) the othercomponent may be a lipase lipase (E.C. 3.1.1.3), a lipid acyltransferase(generally classified as E.C. 2.3.1.x), or a phospholipase (E.C.3.1.1.4, E.C. 3.1.1.32 or E.C. 3.1.1.5), suitably a lipase (E.C.3.1.1.3).

In one embodiment (particularly for feed applications) the othercomponent may be a protease (e.g. subtilisin (E.C. 3.4.21.62) or abacillolysin (E.C. 3.4.24.28) or an alkaline serine protease (E.C.3.4.21.x) or a keratinase (E.C. 3.4.x.x)).

In one embodiment the additional component may be a stabiliser or anemulsifier or a binder or carrier or an excipient or a diluent or adisintegrant.

The term “stabiliser” as used here is defined as an ingredient orcombination of ingredients that keeps a product (e.g. a feed product)from changing over time.

The term “emulsifier” as used herein refers to an ingredient (e.g. afeed ingredient) that prevents the separation of emulsions. Emulsionsare two immiscible substances, one present in droplet form, containedwithin the other. Emulsions can consist of oil-in-water, where thedroplet or dispersed phase is oil and the continuous phase is water, orwater-in-oil, where the water becomes the dispersed phase and thecontinuous phase is oil. Foams, which are gas-in-liquid, andsuspensions, which are solid-in-liquid, can also be stabilised throughthe use of emulsifiers.

As used herein the term “binder” refers to an ingredient (e.g. a feedingredient) that binds the product together through a physical orchemical reaction. During “gelation” for instance, water is absorbed,providing a binding effect. However, binders can absorb other liquids,such as oils, holding them within the product. In the context of thepresent invention binders would typically be used in solid orlow-moisture products for instance baking products: pastries, doughnuts,bread and others. Examples of granulation binders include one or moreof: polyvinylpyrrolidone, hydroxypropylmethylcellulose (HPMC),hydroxypropylcellulose (HPC), sucrose, maltose, gelatin and acacia.

“Carriers” mean materials suitable for administration of the enzyme andinclude any such material known in the art such as, for example, anyliquid, gel, solvent, liquid diluent, solubilizer, or the like, which isnon-toxic and which does not interact with any components of thecomposition in a deleterious manner.

The present invention provides a method for preparing a composition(e.g. a feed additive composition) comprising admixing an enzyme of thepresent invention with at least one physiologically acceptable carrierselected from at least one of maltodextrin, limestone (calciumcarbonate), cyclodextrin, wheat or a wheat component, sucrose, starch,Na₂SO₄, Talc, PVA, sorbitol, benzoate, sorbiate, glycerol, sucrose,propylene glycol, 1,3-propane diol, glucose, parabens, sodium chloride,citrate, acetate, phosphate, calcium, metabisulfite, formate andmixtures thereof.

Examples of “excipients” include one or more of: microcrystallinecellulose and other celluloses, lactose, sodium citrate, calciumcarbonate, dibasic calcium phosphate, glycine, starch, milk sugar andhigh molecular weight polyethylene glycols.

Examples of “disintegrants” include one or more of: starch (preferablycorn, potato or tapioca starch), sodium starch glycollate,croscarmellose sodium and certain complex silicates.

Examples of “diluents” include one or more of: water, ethanol, propyleneglycol and glycerin, and combinations thereof.

The other components may be used simultaneously (e.g. when they are inadmixture together or even when they are delivered by different routes)or sequentially (e.g. they may be delivered by different routes) to thexylanase of the present invention.

Preferably, when the feed additive composition of the present inventionis admixed with another component(s), the DFM remains viable.

In one embodiment preferably the feed additive composition according tothe present invention does not comprise chromium or organic chromium.

In one embodiment preferably the feed additive according to the presentinvention does not contain glucanase.

In one embodiment preferably the feed additive according to the presentinvention does not contain sorbic acid.

Isolated

In one aspect, preferably the amino acid sequence, or nucleic acid, orenzyme according to the present invention is in an isolated form. Theterm “isolated” means that the sequence or enzyme or nucleic acid is atleast substantially free from at least one other component with whichthe sequence, enzyme or nucleic acid is naturally associated in natureand as found in nature. The sequence, enzyme or nucleic acid of thepresent invention may be provided in a form that is substantially freeof one or more contaminants with which the substance might otherwise beassociated. Thus, for example it may be substantially free of one ormore potentially contaminating polypeptides and/or nucleic acidmolecules.

Purified

In one aspect, preferably the sequence, enzyme or nucleic acid accordingto the present invention is in a purified form. The term “purified”means that the given component is present at a high level. The componentis desirably the predominant component present in a composition.Preferably, it is present at a level of at feast about 90%, or at leastabout 95% or at least about 98%, said level being determined on a dryweight/dry weight basis with respect to the total composition underconsideration.

Nucleotide Sequence

The scope of the present invention encompasses nucleotide sequencesencoding proteins having the specific properties as defined herein.

The term “nucleotide sequence” as used herein refers to anoligonucleotide sequence or polynucleotide sequence, and variant,homologues, fragments and derivatives thereof (such as portionsthereof). The nucleotide sequence may be of genomic or synthetic orrecombinant origin, which may be double-stranded or single-strandedwhether representing the sense or anti-sense strand.

The term “nucleotide sequence” in relation to the present inventionincludes genomic DNA, cDNA, synthetic DNA, and RNA. Preferably it meansDNA, more preferably cDNA sequence coding for the present invention.

In a preferred embodiment, the nucleotide sequence when relating to andwhen encompassed by the per se scope of the present invention does notinclude the native nucleotide sequence according to the presentinvention when in its natural environment and when it is linked to itsnaturally associated sequence(s) that is/are also in its/their naturalenvironment. For ease of reference, we shall call this preferredembodiment the “non-native nucleotide sequence”. In this regard, theterm “native nucleotide sequence” means an entire nucleotide sequencethat is in its native environment and when operatively linked to anentire promoter with which it is naturally associated, which promoter isalso in its native environment. However, the amino acid sequenceencompassed by scope the present invention can be isolated and/orpurified post expression of a nucleotide sequence in its nativeorganism. Preferably, however, the amino acid sequence encompassed byscope of the present invention may be expressed by a nucleotide sequencein its native organism but wherein the nucleotide sequence is not underthe control of the promoter with which it is naturally associated withinthat organism.

Typically, the nucleotide sequence encompassed by the scope of thepresent invention is prepared using recombinant DNA techniques (i.e.recombinant DNA). However, in an alternative embodiment of theinvention, the nucleotide sequence could be synthesised, in whole or inpart, using chemical methods well known in the art (see Caruthers M H etal., (1980) Nuc Acids Res Symp Ser 215-23 and Horn T et al, (1980) NucAcids Res Symp Ser 225-232).

Preparation of the Nucleotide Sequence

A nucleotide sequence encoding either a protein which has the specificproperties as defined herein or a protein which is suitable formodification may be identified and/or isolated and/or purified from anycell or organism producing said protein. Various methods are well knownwithin the art for the identification and/or isolation and/orpurification of nucleotide sequences. By way of example, PCRamplification techniques to prepare more of a sequence may be used oncea suitable sequence has been identified and/or isolated and/or purified.

By way of further example, a genomic DNA and/or cDNA library may beconstructed using chromosomal DNA or messenger RNA from the organismproducing the enzyme. If the amino acid sequence of the enzyme is known,labelled oligonucleotide probes may be synthesised and used to identifyenzyme-encoding clones from the genomic library prepared from theorganism. Alternatively, a labelled oligonucleotide probe containingsequences homologous to another known enzyme gene could be used toidentify enzyme-encoding clones. In the latter case, hybridisation andwashing conditions of lower stringency are used.

Alternatively, enzyme-encoding clones could be identified by insertingfragments of genomic DNA into an expression vector, such as a plasmid,transforming enzyme-negative bacteria with the resulting genomic DNAlibrary, and then plating the transformed bacteria onto agar platescontaining a substrate for enzyme (i.e. maltose), thereby allowingclones expressing the enzyme to be identified.

In a yet further alternative, the nucleotide sequence encoding theenzyme may be prepared synthetically by established standard methods,e.g. the phosphoroamidite method described by Beucage S. L. et al.,(1981) Tetrahedron Letters 22, p 1859-1869, or the method described byMatthes et al., (1984) EMBO J. 3, p 801-805. In the phosphoroamiditemethod, oligonucleotides are synthesised, e.g. in an automatic DNAsynthesiser, purified, annealed, ligated and cloned in appropriatevectors.

The nucleotide sequence may be of mixed genomic and synthetic origin,mixed synthetic and cDNA origin, or mixed genomic and cDNA origin,prepared by ligating fragments of synthetic, genomic or cDNA origin (asappropriate) in accordance with standard techniques. Each ligatedfragment corresponds to various parts of the entire nucleotide sequence.The DNA sequence may also be prepared by polymerase chain reaction (PCR)using specific primers, for instance as described in U.S. Pat. No.4,683,202 or in Saiki R K et al., (Science (1988) 239, pp 487-491).

Amino Acid Sequences

The scope of the present invention also encompasses amino acid sequencesof enzymes having the specific properties as defined herein.

As used herein, the term “amino acid sequence” is synonymous with theterm “polypeptide” and/or the term “protein”. In some instances, theterm “amino acid sequence” is synonymous with the term “peptide”. Insome instances, the term “amino acid sequence” is synonymous with theterm “enzyme”.

The amino acid sequence may be prepared/isolated from a suitable source,or it may be made synthetically or it may be prepared by use ofrecombinant DNA techniques.

Preferably the amino acid sequence when relating to and when encompassedby the per se scope of the present invention is not a native enzyme. Inthis regard, the term “native enzyme” means an entire enzyme that is inits native environment and when it has been expressed by its nativenucleotide sequence.

Sequence Identity or Sequence Homology

The present invention also encompasses the use of sequences having adegree of sequence identity or sequence homology with amino acidsequence(s) of a polypeptide having the specific properties definedherein or of any nucleotide sequence encoding such a polypeptide(hereinafter referred to as a “homologous sequence(s)”). Here, the term“homologue” means an entity having a certain homology with the subjectamino acid sequences and the subject nucleotide sequences. Here, theterm “homology” can be equated with “identity”.

The homologous amino acid sequence and/or nucleotide sequence shouldprovide and/or encode a polypeptide which retains the functionalactivity and/or enhances the activity of the enzyme.

In the present context, in some embodiments a homologous sequence istaken to include an amino acid or a nucleotide sequence which may be atleast 97.7% identical, preferably at least 98 or 99% identical to thesubject sequence.

In some embodiments a homologous sequence is taken to include an aminoacid or a nucleotide sequence which may be at least 85% identical,preferably at least 90 or 95% identical to the subject sequence.

Typically, the homologues will comprise the same active sites etc. asthe subject amino acid sequence for instance. Although homology can alsobe considered in terms of similarity (i.e. amino acid residues havingsimilar chemical properties/functions), in the context of the presentinvention it is preferred to express homology in terms of sequenceidentity.

In one embodiment, a homologous sequence is taken to include an aminoacid sequence or nucleotide sequence which has one or several additions,deletions and/or substitutions compared with the subject sequence.

In the present context, “the subject sequence” relates to the nucleotidesequence or polypeptide/amino acid sequence according to the invention.

Preferably, the % sequence identity with regard to a polypeptidesequence is determined using SEQ ID No. 3 as the subject sequence in asequence alignment. In one embodiment, the polypeptide subject sequenceis selected from the group consisting of SEQ ID No. 3, SEQ ID No. 1, SEQID No. 2, SEQ ID No. 9, SEQ ID No. 10, SEQ ID No. 11 or SEQ ID No. 15.In a preferred embodiment the polypeptide subject sequence is selectedfrom the mature sequences SEQ ID No. 3, SEQ ID No. 11 or SEQ ID No. 15.

Preferably, the % sequence identity with regard to a nucleotide sequenceis determined using SEQ ID No. 6 as the subject sequence in the sequencealignment. In one embodiment, the subject sequence for nucleotidesequences may be selected from the group consisting of SEQ ID No. 4, SEQID No. 5, SEQ ID No. 6, SEQ ID No. 12, SEQ ID No. 13, SEQ ID No. 14, SEQID No. 16, SEQ ID No. 17 and SEQ ID No. 18. In a preferred embodimentthe subject sequence is sequence SEQ ID No. 6.

A “parent nucleic acid” or “parent amino acid” means a nucleic acidsequence or amino acid sequence, encoding or coding for the parentpolypeptide, respectively.

In one embodiment the present invention relates to a protein whose aminoacid sequence is represented herein or a protein derived from this(parent) protein by substitution, deletion or addition of one or severalamino acids, such as 2, 3, 4, 5, 6, 7, 8, 9 amino acids, or more aminoacids, such as 10 or more than 10 amino acids in the amino acid sequenceof the parent protein and having the activity of the parent protein.

Suitably, the degree of identity with regard to an amino acid sequenceis determined over at least 20 contiguous amino acids, preferably overat least 30 contiguous amino acids, preferably over at least 40contiguous amino acids, preferably over at least 50 contiguous aminoacids, preferably over at least 60 contiguous amino acids, preferablyover at least 100 contiguous amino acids, preferably over at least 200contiguous amino acids.

In one embodiment the present invention relates to a nucleic acidsequence (or gene) encoding a protein whose amino acid sequence isrepresented herein or encoding a protein derived from this (parent)protein by substitution, deletion or addition of one or several aminoacids, such as 2, 3, 4, 5, 6, 7, 8, 9 amino acids, or more amino acids,such as 10 or more than 10 amino acids in the amino acid sequence of theparent protein and having the activity of the parent protein.

In the present context, in one embodiment a homologous sequence orforeign sequence is taken to include a nucleotide sequence which may beat least 97.7% identical, preferably at least 98 or 99% identical to anucleotide sequence encoding a polypeptide of the present invention (thesubject sequence).

In another embodiment, a homologous sequence is taken to include anucleotide sequence which may be at least 85% identical, preferably atleast 90 or 95% identical to a nucleotide sequence encoding apolypeptide of the present invention (the subject sequence).

Typically, the homologues will comprise the same sequences that code forthe active sites etc. as the subject sequence. Although homology canalso be considered in terms of similarity (i.e. amino acid residueshaving similar chemical properties/functions), in the context of thepresent invention it is preferred to express homology in terms ofsequence identity.

Homology comparisons can be conducted by eye, or more usually, with theaid of readily available sequence comparison programs. Thesecommercially available computer programs can calculate % homology or %identity between two or more sequences.

% homology or % identity may be calculated over contiguous sequences,i.e. one sequence is aligned with the other sequence and each amino acidin one sequence is directly compared with the corresponding amino acidin the other sequence, one residue at a time. This is called an“ungapped” alignment. Typically, such ungapped alignments are performedonly over a relatively short number of residues.

Although this is a very simple and consistent method, it fails to takeinto consideration that, for example, in an otherwise identical pair ofsequences, one insertion or deletion will cause the following amino acidresidues to be put out of alignment, thus potentially resulting in alarge reduction in % homology or % identity when a global alignment isperformed. Consequently, most sequence comparison methods are designedto produce optimal alignments that take into consideration possibleinsertions and deletions without penalising unduly the overall homologyscore. This is achieved by inserting “gaps” in the sequence alignment totry to maximise local homology.

However, these more complex methods assign “gap penalties” to each gapthat occurs in the alignment so that, for the same number of identicalamino acids, a sequence alignment with as few gaps aspossible—reflecting higher relatedness between the two comparedsequences—will achieve a higher score than one with many gaps. “Affinegap costs” are typically used that charge a relatively high cost for theexistence of a gap and a smaller penalty for each subsequent residue inthe gap. This is the most commonly used gap scoring system. High gappenalties will of course produce optimised alignments with fewer gaps.Most alignment programs allow the gap penalties to be modified. However,it is preferred to use the default values when using such software forsequence comparisons.

Calculation of maximum % homology or % identity therefore firstlyrequires the production of an optimal alignment, taking intoconsideration gap penalties. A suitable computer program for carryingout such an alignment is the Vector NTI (Invitrogen Corp.). Examples ofsoftware that can perform sequence comparisons include, but are notlimited to, the BLAST package (see Ausubel et al 1999 Short Protocols inMolecular Biology, 4th Ed—Chapter 18), BLAST 2 (see FEMS Microbiol Lett1999 174(2): 247-50; FEMS Microbiol Lett 1999 177(1): 187-8 andtatiana@ncbi.nlm.nih.gov). FASTA (Altschul et al 1990 J. Mol. Biol.403-410) and AlignX for example. At least BLAST, BLAST 2 and FASTA areavailable for offline and online searching (see Ausubel et al 1999,pages 7-58 to 7-60), such as for example in the GenomeQuest search tool(www.genomequest.com).

Although the final % homology or % identity can be measured in terms ofidentity, the alignment process itself is typically not based on anall-or-nothing pair comparison. Instead, a scaled similarity scorematrix is generally used that assigns scores to each pairwise comparisonbased on chemical similarity or evolutionary distance. An example ofsuch a matrix commonly used is the BLOSUM62 matrix—the default matrixfor the BLAST suite of programs. Vector NTl programs generally useeither the public default values or a custom symbol comparison table ifsupplied (see user manual for further details). For some applications,it is preferred to use the default values for the Vector NTl package.

Alternatively, percentage homologies may be calculated using themultiple alignment feature in Vector NTl (Invitrogen Corp.), based on analgorithm, analogous to CLUSTAL (Higgins D G & Sharp P M (1988), Gene73(1), 237-244).

Once the software has produced an optimal alignment, it is possible tocalculate % homology, preferably % sequence identity. The softwaretypically does this as part of the sequence comparison and generates anumerical result.

Should Gap Penalties be used when determining sequence identity, thenpreferably the following parameters are used for pairwise alignment:

FOR BLAST GAP OPEN 9 GAP EXTENSION 2

FOR CLUSTAL DNA PROTEIN Weight Matrix IUB Gonnet 250 GAP OPENING 15 10GAP EXTEND 6.66 0.1

In one embodiment, CLUSTAL may be used with the gap penalty and gapextension set as defined above.

Suitably, the degree of identity with regard to a nucleotide sequence orprotein sequence is determined over at least 20 contiguousnucleotides/amino acids, preferably over at least 30 contiguousnucleotides/amino acids, preferably over at least 40 contiguousnucleotides/aminio acids, preferably over at least 50 contiguousnucleotides/amino acids, preferably over at least 60 contiguousnucleotides/amino acids, preferably over at least 100 contiguousnucleotides/amino acids.

Suitably, the degree of identity with regard to a nucleotide sequencesequence is determined over at least 100 contiguous nucleotides,preferably over at least 200 contiguous nucleotides, preferably over atleast 300 contiguous nucleotides, preferably over at least 400contiguous nucleotides, preferably over at least 500 contiguousnucleotides, preferably over at least 600 contiguous nucleotides,preferably over at least 700 contiguous nucleotides, preferably over atleast 800 contiguous nucleotides.

Suitably, the degree of identity with regard to a nucleotide sequencemay be determined over the whole sequence taught herein.

Suitably, the degree of identity with regard to a nucleotide sequencemay be determined over the whole sequence taught herein as the maturesequence, e.g. SEQ ID No. 6 or SEQ ID No. 14 or SEQ ID No. 18. Suitably,the degree of identity with regard to a nucleotide sequence may bedetermined over the whole sequence as taught herein as SEQ ID No. 6.

Suitably, the degree of identity with regard to a protein (amino acid)sequence is determined over at least 100 contiguous amino acids,preferably over at least 200 contiguous amino acids, preferably over atleast 300 contiguous amino acids.

Suitably, the degree of identity with regard to an amino acid or proteinsequence may be determined over the whole sequence taught herein.

Suitably, the degree of identity with regard to an amino acid or proteinsequence may be determined over the whole sequence taught herein as themature sequence, e.g. SEQ ID No. 3, SEQ ID No. 11 or SEQ ID No. 15.Suitably, the degree of identity with regard to an amino acid or proteinsequence may be determined over the whole sequence taught herein as SEQID No. 3.

In the present context, the term “query sequence” means a homologoussequence or a foreign sequence, which is aligned with a subject sequencein order to see if it falls within the scope of the present invention.Accordingly, such query sequence can for example be a prior art sequenceor a third party sequence.

In one preferred embodiment, the sequences are aligned by a globalalignment program and the sequence identity is calculated by identifyingthe number of exact matches identified by the program divided by thelength of the subject sequence.

In one embodiment, the degree of sequence identity between a querysequence and a subject sequence is determined by 1) aligning the twosequences by any suitable alignment program using the default scoringmatrix and default gap penalty, 2) identifying the number of exactmatches, where an exact match is where the alignment program hasidentified an identical amino acid or nucleotide in the two alignedsequences on a given position in the alignment and 3) dividing thenumber of exact matches with the length of the subject sequence.

In yet a further preferred embodiment, the global alignment program isselected from the group consisting of CLUSTAL and BLAST (preferablyBLAST) and the sequence identity is calculated by identifying the numberof exact matches identified by the program divided by the length of thesubject sequence.

The sequences may also have deletions, insertions or substitutions ofamino acid residues result in a functionally equivalent substance.Deliberate amino acid substitutions may be made on the basis ofsimilarity in polarity, charge, solubility, hydrophobicity,hydrophilicity, and/or the amphipathic nature of the residues. Forexample, negatively charged amino acids include aspartic acid andglutamic acid; positively charged amino acids include lysine andarginine; and amino acids with uncharged polar head groups havingsimilar hydrophilicity values include leucine, isoleucine, valine,glycine, alanine, asparagine, glutamine, serine, threonine,phenylalanine, and tyrosine.

Conservative substitutions may be made, for example according to theTable below. Amino acids in the same block in the second column andpreferably in the same line in the third column may be substituted foreach other:

ALIPHATIC Non-polar G A P I L V Polar - uncharged C S T M N Q Polar -charged D E K R AROMATIC H F W Y

The present invention also encompasses homologous substitution(substitution and replacement are both used herein to mean theinterchange of an existing amino acid residue, with an alternativeresidue) that may occur i.e. like-for-like substitution such as basicfor basic, acidic for acidic, polar for polar etc. Non-homologoussubstitution may also occur i.e. from one class of residue to another oralternatively involving the inclusion of unnatural amino acids such asornithine (hereinafter referred to as Z), diaminobutyric acid ornithine(hereinafter referred to as B), norleucine ornithine (hereinafterreferred to as O), pyriylalanine, thienylalanine, naphthylalanine andphenylglycine.

Replacements may also be made by unnatural amino acids include; alpha*and alpha-disubstituted* amino acids, N-alkyl amino acids*, lacticacid*, halide derivatives of natural amino acids such astrifluorotyrosine*, p-Cl-phenylalanine* p-Br-phenylalanine*,p-I-phenylalanine*, L-allyl-glycine*, β-alanine*, L-α-amino butyricacid*, L-γ-amino butyric acid*, L-α-amino isobutyric acid*, L-ϵ-aminocaproic acid#, 7-amino heptanoic acid*, L-methionine sulfone#*,L-norleucine*, L-norvaline*, p-nitro-L-phenylalanine*, L-hydroxyproline*L-thioproline*, methyl derivatives of phenylalanine (Phe) such as4-methyl-Phe*, pentamethyl-Phe*, L-Phe (4-amino)#, L-Tyr (methyl)*,L-Phe (4-isopropyl)*, L-Tic (1,2,3,4-tetrahydroisoquinoline-3-carboxylacid)*, L-diaminopropionic acid# and L-Phe (4-benzyl)*. The notation *has been utilised for the purpose of the discussion above (relating tohomologous or non-homologous substitution), to indicate the hydrophobicnature of the derivative whereas # has been utilised to indicate thehydrophilic nature of the derivative, #* indicates amphipathiccharacteristics.

Variant amino acid sequences may include suitable spacer groups that maybe inserted between any two amino acid residues of the sequenceincluding alkyl groups such as methyl, ethyl or propyl groups inaddition to amino acid spacers such as glycine or β-alanine residues. Afurther form of variation, involves the presence of one or more aminoacid residues in peptoid form, will be well understood by those skilledin the art. For the avoidance of doubt, “the peptoid form” is used torefer to variant amino acid residues wherein the α-carbon substituentgroup is on the residue's nitrogen atom rather than the α-carbon.Processes for preparing peptides in the peptoid form are known in theart, for example Simon R J et al., PNAS (1992) 89(20), 9367-9371 andHorwell D C, Trends Biotechnol. (1995) 13(4), 132-134.

In one embodiment the xylanase for use in the present invention maycomprise a polypeptide sequence shown as SEQ ID No. 1, SEQ ID No. 2 orSEQ ID No. 3, SEQ ID No. 9, SEQ ID No. 10 SEQ ID No. 11 or SEQ ID No. 15with a conservative substitution of at least one of the amino acids.

Suitably there may be at least 2 conservative substitutions, such as atleast 3 or at least 4 or at least 5.

Suitably there may be less than 15 conservative substitutions, such asless than 12, less than 10, or less than 8 or less than 5.

The nucleotide sequences for use in the present invention may includewithin them synthetic or modified nucleotides. A number of differenttypes of modification to oligonucleotides are known in the art. Theseinclude methylphosphonate and phosphorothioate backbones and/or theaddition of acridine or polylysine chains at the 3′ and/or 5′ ends ofthe molecule. For the purposes of the present invention, it is to beunderstood that the nucleotide sequences described herein may bemodified by any method available in the art. Such modifications may becarried out in order to enhance the in vivo activity or life span ofnucleotide sequences of the present invention.

The present invention also encompasses the use of nucleotide sequencesthat are complementary to the sequences presented herein, or anyderivative, fragment or derivative thereof. If the sequence iscomplementary to a fragment thereof then that sequence can be used as aprobe to identify similar coding sequences in other organisms etc.

Polynucleotides which are not 100% homologous to the sequences of thepresent invention but fall within the scope of the invention can beobtained in a number of ways. Other variants of the sequences describedherein may be obtained for example by probing DNA libraries made from arange of individuals, for example individuals from differentpopulations. In addition, other homologues may be obtained and suchhomologues and fragments thereof in general will be capable ofselectively hybridising to the sequences shown in the sequence listingherein. Such sequences may be obtained by probing cDNA libraries madefrom or genomic DNA libraries from other animal species, and probingsuch libraries with probes comprising all or part of any one of thesequences in the attached sequence listings under conditions of mediumto high stringency. Similar considerations apply to obtaining specieshomologues and allelic variants of the polypeptide or nucleotidesequences of the invention.

Variants and strain/species homologues may also be obtained usingdegenerate PCR which will use primers designed to target sequenceswithin the variants and homologues encoding conserved amino acidsequences within the sequences of the present invention. Conservedsequences can be predicted, for example, by aligning the amino acidsequences from several variants/homologues. Sequence alignments can beperformed using computer software known in the art. For example the GCGWisconsin PileUp program is widely used.

The primers used in degenerate PCR will contain one or more degeneratepositions and will be used at stringency conditions lower than thoseused for cloning sequences with single sequence primers against knownsequences.

Alternatively, such polynucleotides may be obtained by site directedmutagenesis of characterised sequences. This may be useful where forexample silent codon sequence changes are required to optimise codonpreferences for a particular host cell in which the polynucleotidesequences are being expressed. Other sequence changes may be desired inorder to introduce restriction enzyme recognition sites, or to alter theproperty or function of the polypeptides encoded by the polynucleotides.

Polynucleotides (nucleotide sequences) of the invention may be used toproduce a primer, e.g. a PCR primer, a primer for an alternativeamplification reaction, a probe e.g. labelled with a revealing label byconventional means using radioactive or non-radioactive labels, or thepolynucleotides, may be cloned into vectors. Such primers, probes andother fragments will be at least 15, preferably at least 20, for exampleat least 25, 30 or 40 nucleotides in length, and are also encompassed bythe term polynucleotides of the invention as used herein.

Polynucleotides such as DNA polynucleotides and probes according to theinvention may be produced recombinantly, synthetically, or by any meansavailable to those of skill in the art. They may also be cloned bystandard techniques.

In general, primers will be produced by synthetic means, involving astepwise manufacture of the desired nucleic acid sequence one nucleotideat a time. Techniques for accomplishing this using automated techniquesare readily available in the art.

Longer polynucleotides will generally be produced using recombinantmeans, for example using, a PCR (polymerase chain reaction) cloningtechniques. The primers may be designed to contain suitable restrictionenzyme recognition sites so that the amplified DNA can be cloned into asuitable cloning vector.

Amino Acid Numbering

In the present invention, a specific numbering of amino acid residuepositions in the xylanases used in the present invention may beemployed. By alignment of the amino acid sequence of a sample xylanaseswith the xylanase of the present invention (particularly SEQ ID No. 3)it is possible to allot a number to an amino acid residue position insaid sample xylanase which corresponds with the amino acid residueposition or numbering of the amino acid sequence shown in SEQ ID NO: 3of the present invention.

Hybridisation

The present invention also encompasses sequences that are complementaryto the nucleic acid sequences of the present invention or sequences thatare capable of hybridising either to the sequences of the presentinvention or to sequences that are complementary thereto.

The term “hybridisation” as used herein shall include “the process bywhich a strand of nucleic acid joins with a complementary strand throughbase pairing” as well as the process of amplification as carried out inpolymerase chain reaction (PCR) technologies.

The present invention also encompasses the use of nucleotide sequencesthat are capable of hybridising to the sequences that are complementaryto the sequences presented herein, or any fragment or derivativethereof.

The term “variant” also encompasses sequences that are complementary tosequences that are capable of hybridising to the nucleotide sequencespresented herein.

Preferably, the term “variant” encompasses sequences that arecomplementary to sequences that are capable of hybridising understringent conditions (e.g. 50° C. and 0.2×SSC {1×SSC=0.15 M NaCl, 0.015M Na₃citrate pH 7.0}) to the nucleotide sequences presented herein.

More preferably, the term “variant” encompasses sequences that arecomplementary to sequences that are capable of hybridising under highstringency conditions (e.g. 65° C. and 0.1×SSC {1×SSC=0.15 M NaCl, 0.015M Na₃citrate pH 7.0}) to the nucleotide sequences presented herein.

The present invention also relates to nucleotide sequences that canhybridise to the nucleotide sequences of the present invention(including complementary sequences of those presented herein).

The present invention also relates to nucleotide sequences that arecomplementary to sequences that can hybridise to the nucleotidesequences of the present invention (including complementary sequences ofthose presented herein).

Preferably hybridisation is analysed over the whole of the sequencestaught herein.

Expression of Enzymes

The nucleotide sequence for use In the present invention may beincorporated into a recombinant replicable vector. The vector may beused to replicate and express the nucleotide sequence, in protein/enzymeform, in and/or from a compatible host cell.

Expression may be controlled using control sequences e.g. regulatorysequences.

The protein produced by a host recombinant cell by expression of thenucleotide sequence may be secreted or may be contained intracellularlydepending on the sequence and/or the vector used. The coding sequencesmay be designed with signal sequences which direct secretion of thesubstance coding sequences through a particular prokaryotic oreukaryotic cell membrane.

Expression Vector

The term “expression vector” means a construct capable of in vivo or invitro expression.

Preferably, the expression vector is incorporated into the genome of asuitable host organism. The term “incorporated” preferably covers stableincorporation into the genome.

The nucleotide sequence of the present invention may be present in avector in which the nucleotide sequence is operably linked to regulatorysequences capable of providing for the expression of the nucleotidesequence by a suitable host organism.

The vectors for use in the present invention may be transformed into asuitable host cell as described below to provide for expression of apolypeptide of the present invention.

The choice of vector e.g. a plasmid, cosmid, or phage vector will oftendepend on the host cell into which it is to be introduced.

The vectors for use in the present invention may contain one or moreselectable marker genes—such as a gene, which confers antibioticresistance e.g. ampicillin, kanamycin, chloramphenicol or tetracyclinresistance. Alternatively, the selection may be accomplished byco-transformation (as described in WO91/17243).

Vectors may be used in vitro, for example for the production of RNA orused to transfect, transform, transduce or infect a host cell.

Thus, in a further embodiment, the invention provides a method of makingnucleotide sequences of the present invention by introducing anucleotide sequence of the present invention into a replicable vector,introducing the vector into a compatible host cell, and growing the hostcell under conditions which bring about replication of the vector.

The vector may further comprise a nucleotide sequence enabling thevector to replicate in the host cell in question. Examples of suchsequences are the origins of replication of plasmids pUC19, pACYC177,pUB110, pE194, pAMB1 and pIJ702.

Regulatory Sequences

In some applications, the nucleotide sequence for use in the presentinvention is operably linked to a regulatory sequence which is capableof providing for the expression of the nucleotide sequence, such as bythe chosen host cell. By way of example, the present invention covers avector comprising the nucleotide sequence of the present inventionoperably linked to such a regulatory sequence, i.e. the vector is anexpression vector.

The term “operably linked” refers to a juxtaposition wherein thecomponents described are in a relationship permitting them to functionin their intended manner. A regulatory sequence “operably linked” to acoding sequence is ligated in such a way that expression of the codingsequence is achieved under condition compatible with the controlsequences.

The term “regulatory sequences” includes promoters and enhancers andother expression regulation signals.

The term “promoter” is used in the normal sense of the art, e.g. an RNApolymerase binding site.

Enhanced expression of the nucleotide sequence encoding the enzyme ofthe present invention may also be achieved by the selection ofheterologous regulatory regions, e.g. promoter, secretion leader andterminator regions.

Preferably, the nucleotide sequence according to the present inventionis operably linked to at least a promoter.

Other promoters may even be used to direct expression of the polypeptideof the present invention.

Examples of suitable promoters for directing the transcription of thenucleotide sequence in a bacterial, fungal or yeast host are well knownin the art.

The promoter can additionally include features to ensure or to increaseexpression in a suitable host. For example, the features can beconserved regions such as a Pribnow Box or a TATA box.

Constructs

The term “construct”—which is synonymous with terms such as “conjugate”,“cassette” and “hybrid”—includes a nucleotide sequence for use accordingto the present invention directly or indirectly attached to a promoter.

An example of an indirect attachment is the provision of a suitablespacer group such as an intron sequence, such as the Sh1-intron or theADH intron, intermediate the promoter and the nucleotide sequence of thepresent invention. The same is true for the term “fused” in relation tothe present invention which includes direct or indirect attachment. Insome cases, the terms do not cover the natural combination of thenucleotide sequence coding for the protein ordinarily associated withthe wild type gene promoter and when they are both in their naturalenvironment.

The construct may even contain or express a marker, which allows for theselection of the genetic construct.

For some applications, preferably the construct of the present inventioncomprises at least the nucleotide sequence of the present inventionoperably linked to a promoter.

Host Cells

The term “host cell”—in relation to the present invention includes anycell that comprises either the nucleotide sequence or an expressionvector as described above and which is used in the recombinantproduction of a protein having the specific properties as definedherein.

In one embodiment the organism is an expression host.

Thus, a further embodiment of the present Invention provides host cellstransformed or transfected with a nucleotide sequence that expresses theprotein of the present invention. The cells will be chosen to becompatible with the said vector and may for example be prokaryotic (forexample bacterial), fungal or yeast cells.

Examples of suitable bacterial host organisms are gram positive or gramnegative bacterial species.

In one embodiment the xylanases taught herein are expressed in theexpression host Trichoderma reesei.

In some embodiments the expression host for the xylanases taught hereinmay be one or more of the following fungal expression hosts: Fusariumspp. (such as Fusarium oxysporum); Aspergillus spp. (such as Aspergillusniger, A. oryzae, A. nidulans, or A. awamori) or Trichoderma spp. (suchas T. reesei).

In some embodiments the expression host may be one or more of thefollowing bacterial expression hosts: Streptomyces spp. or Bacillus spp.(e.g. Bacillus subtilis or B. licheniformis).

The use of suitable host cells—such as yeast and fungal host cells—mayprovide for post-translational modifications (e.g. myristoylation,glycosylation, truncation, lipidation and tyrosine, serine or threoninephosphorylation) as may be needed to confer optimal biological activityon recombinant expression products of the present invention.

Organism

The term “organism” in relation to the present invention includes anyorganism that could comprise the nucleotide sequence coding for thepolypeptide according to the present invention and/or products obtainedtherefrom, and/or wherein a promoter can allow expression of thenucleotide sequence according to the present invention when present inthe organism.

In one embodiment the organism is an expression host.

Suitable organisms may include a prokaryote, fungus, yeast or a plant.

The term “transgenic organism” in relation to the present inventionincludes any organism that comprises the nucleotide sequence coding forthe polypeptide according to the present invention and/or the productsobtained therefrom, and/or wherein a promoter can allow expression ofthe nucleotide sequence according to the present invention within theorganism. Preferably the nucleotide sequence is incorporated in thegenome of the organism.

The term “transgenic organism” does not cover native nucleotide codingsequences in their natural environment when they are under the controlof their native promoter which is also in its natural environment.

Therefore, the transgenic organism of the present invention includes anorganism comprising any one of, or combinations of, the nucleotidesequence coding for the polypeptide according to the present invention,constructs according to the present invention, vectors according to thepresent invention, plasmids according to the present invention, cellsaccording to the present invention, tissues according to the presentinvention, or the products thereof.

For example the transgenic organism may also comprise the nucleotidesequence coding for the polypeptide of the present invention under thecontrol of a heterologous promoter.

Transformation of Host Cells/Organism

As indicated earlier, the host organism can be a prokaryotic or aeukaryotic organism. Examples of suitable prokaryotic hosts include E.coli, Streptomyces spp. and Bacillus spp., e.g. Bacillus subtilis.

Teachings on the transformation of prokaryotic hosts is well documentedin the art, for example see Sambrook et al (Molecular Cloning: ALaboratory Manual, 2nd edition, 1989, Cold Spring Harbor LaboratoryPress). If a prokaryotic host is used then the nucleotide sequence mayneed to be suitably modified before transformation—such as by removal ofintrons.

Filamentous fungi cells may be transformed using various methods knownin the art—such as a process involving protoplast formation andtransformation of the protoplasts followed by regeneration of the cellwall in a manner known. The use of Aspergillus as a host microorganismis described in EP 0 238 023.

Transformation of prokaryotes, fungi and yeasts are generally well knownto one skilled in the art.

A host organism may be a fungus—such as a mould. Examples of suitablesuch hosts include any member belonging to the genera Trichoderma (e.g.T. reesei), Thermomyces, Acremonium, Fusarium, Aspergillus, Penicillium,Mucor, Neurospora and the like.

In one embodiment, the host organism may be a fungus. In one preferredembodiment the host organism belongs to the genus Trichoderma, e.g. T.reesei).

Cultur1ng and Production

Host cells transformed with the nucleotide sequence of the presentinvention may be cultured under conditions conducive to the productionof the encoded polypeptide and which facilitate recovery of thepolypeptide from the cells and/or culture medium.

The medium used to cultivate the cells may be any conventional mediumsuitable for growing the host cell in questions and obtaining expressionof the polypeptide.

The protein produced by a recombinant cell may be displayed on thesurface of the cell.

The protein may be secreted from the host cells and may conveniently berecovered from the culture medium using well-known procedures.

Secretion

Often, it is desirable for the protein to be secreted from theexpression host into the culture medium from where the protein may bemore easily recovered. According to the present invention, the secretionleader sequence may be selected on the basis of the desired expressionhost. Hybrid signal sequences may also be used with the context of thepresent invention.

Large Scale Application

In one preferred embodiment of the present invention, the amino acidsequence is used for large scale applications.

Preferably the amino acid sequence is produced in a quantity of from 1 gper litre to about 2 g per litre of the total cell culture volume aftercultivation of the host organism.

Preferably the amino acid sequence is produced in a quantity of from 100mg per litre to about 900 mg per litre of the total cell culture volumeafter cultivation of the host organism.

Preferably the amino acid sequence is produced in a quantity of from 250mg per litre to about 500 mg per litre of the total cell culture volumeafter cultivation of the host organism.

General Recombinant DNA Methodology Techniques

The present invention employs, unless otherwise indicated, conventionaltechniques of chemistry, molecular biology, microbiology, recombinantDNA and immunology, which are within the capabilities of a person ofordinary skill in the art. Such techniques are explained in theliterature. See, for example, J. Sambrook, E. F. Fritsch, and T.Maniatis, 1989, Molecular Cloning: A Laboratory Manual, Second Edition,Books 1-3, Cold Spring Harbor Laboratory Press; Ausubel, F. M. et al.(1995 and periodic supplements; Current Protocols in Molecular Biology,ch. 9, 13, and 16, John Wiley & Sons, New York, N.Y.); B. Roe, J.Crabtree, and A. Kahn, 1996, DNA Isolation and Sequencing: EssentialTechniques, John Wiley & Sons; M. J. Gait (Editor), 1984,Oligonucleotide Synthesis: A Practical Approach, Irl Press; and, D. M.J. Lilley and J. E. Dahlberg, 1992, Methods of Enzymology: DNA StructurePart A: Synthesis and Physical Analysis of DNA Methods in Enzymology,Academic Press. Each of these general texts is herein incorporated byreference.

The invention also relates to the following aspects as defined in thefollowing numbered paragraphs:

-   -   1. A method for degrading arabinoxylan-containing material        comprising admixing an arabinoxylan-containing material with a        xylanase, which xylanase is a GH10, fungal xylanase and degrades        insoluble arabinoxylan (AXinsol) as well as degrading the        polymers, oligomers or combinations thereof produced from the        degradation of the AXinsol, and wherein the xylanase degrades        the polymers, oligomers or combinations thereof produced from        the degradation of the AXinsol immediately or substantially        immediately upon their production.    -   2. A method for degrading arabinoxylan-containing material in a        xylan-containing material, comprising admixing said        xylan-containing material with a xylanase comprising a        polypeptide sequence shown herein as SEQ ID No. 3, SEQ ID No. 2,        SEQ ID No. 1, SEQ ID No. 9, SEQ ID No. 10, SEQ ID No. 11 of SEQ        ID No. 15; or a variant, homologue, fragment or derivative        thereof having at least 75% identity with SEQ ID No. 3 or SEQ ID        No. 2 or SEQ ID No. 1 or SEQ ID No. 9 or SEQ ID No. 10 or SEQ ID        No. 11 or SEQ ID No. 15; or a polypeptide sequence which        comprises SEQ ID No. 3, SEQ ID No. 2, SEQ ID No. 1, SEQ ID No.        9, SEQ ID No. 10, SEQ ID No. 11 or SEQ ID No. 15 with a        conservative substitution of at least one of the amino acids; or        a xylanase which is encoded by a nucleotide sequence shown        herein as SEQ ID No. 6, SEQ ID No. 5, SEQ ID No. 4, SEQ ID No.        12, SEQ ID No. 13, SEQ ID No. 14, SEQ ID No. 16, SEQ ID No. 17        or SEQ ID No. 18, or a nucleotide sequence which can hybridize        to SEQ ID No. 6, SEQ ID No. 5, SEQ ID No. 4, SEQ ID No. 12, SEQ        ID No. 13, SEQ ID No. 14, SEQ ID No. 16, SEQ ID No. 17 or SEQ ID        No. 18 under high stringency conditions, or a nucleotide        sequence which has at least 75% identity with SEQ ID No. 6, SEQ        ID No. 5, SEQ ID No. 4, SEQ ID No. 12, SEQ ID No. 13, SEQ ID No.        14, SEQ ID No. 16, SEQ ID No. 17 or SEQ ID No. 18, or a        nucleotide sequence which differs from SEQ ID No. 6 or SEQ ID        No. 5 or SEQ ID No. 4 or SEQ ID No. 12 or SEQ ID No. 13 or SEQ        ID No. 14 or SEQ ID No. 16 or SEQ ID No. 17 or SEQ ID No. 18 due        to the degeneracy of the genetic code, or a xylanase obtainable        (or obtained) from Fusarium verticilloides.    -   3. Use of a xylanase comprising a polypeptide sequence shown        herein as SEQ ID No. 3, SEQ ID No. 2, SEQ ID No. 1, SEQ ID No.        9, SEQ ID No. 10, SEQ ID No. 11, or or SEQ ID No. 15; or a        variant, homologue, fragment or derivative thereof having at        least 75% identity with SEQ ID No. 3 or SEQ ID No. 2 or SEQ ID        No. 1 or SEQ ID No. 9 or SEQ ID No. 10 or SEQ ID No. 11 or SEQ        ID No. 15; or a polypeptide sequence which comprises SEQ ID No.        3, SEQ ID No. 2, SEQ ID No. 1, SEQ ID No. 9, SEQ ID No. 10, SEQ        ID No. 11 or SEQ ID No. 15 with a conservative substitution of        at least one of the amino acids; or a xylanase which is encoded        by a nucleotide sequence shown herein as SEQ ID No. 6, SEQ ID        No. 5, SEQ ID No. 4, SEQ ID No. 12, SEQ ID No. 13, SEQ ID No.        14, SEQ ID No. 16, SEQ ID No. 17 or SEQ ID No. 18, or a        nucleotide sequence which can hybridize to SEQ ID No. 6, SEQ ID        No. 5, SEQ ID No. 4, SEQ ID No. 12, SEQ ID No. 13, SEQ ID No.        14, SEQ ID No. 16, SEQ ID No. 17 or SEQ ID No. 18 under high        stringency conditions, or a nucleotide sequence which has at        least 75% identity with SEQ ID No. 6, SEQ ID No. 5, SEQ ID No.        4, SEQ ID No. 12, SEQ ID No. 13, SEQ ID No. 14, SEQ ID No. 16,        SEQ ID No. 17 or SEQ ID No. 18, or a nucleotide sequence which        differs from SEQ ID No. 6 or SEQ ID No. 5 or SEQ ID No. 4 or SEQ        ID No. 12 or SEQ ID No. 13 or SEQ ID No. 14 or SEQ ID No. 16 or        SEQ ID No. 17 or SEQ ID No. 18 due to the degeneracy of the        genetic code, or a xylanase obtainable (or obtained) from        Fusarium verticilloides for solubilizing arabinoxylan in a        xylan-containing material.    -   4. The method or use according to any one of paragraphs 2 or 3        wherein the arabinoxylan is insoluble arabinoxylan (AXinsol).    -   5. The method or use according to any one of paragraphs 1-4        wherein the xylan-containing material is selected from one or        more of the group consisting of: a feed or feedstuff; a feed        component; a grain-based material; a mash; a wort; a malt;        malted barley; an adjunct, a barley mash; and a cereal flour.    -   6. The method or use according to any one of the preceding        paragraphs wherein the arabinoxylans are solubilized without        increasing viscosity in the reaction medium.    -   7. The method or use according to any one of the preceding        paragraphs wherein the method or use is (or is part of) a wheat        gluten-starch separation process.    -   8. The method or use according to any one of paragraphs 1-6        wherein the method or use is (or is part of) a biofuel (e.g.        bioethanol) or biochemical (e.g. bio-based isoprene) production        process.    -   9. The method or use according to any one of paragraphs 1-6        wherein the method or use is (or is part of) a malting or        brewing process.    -   10. The method or use according to any one of paragraphs 1-6        wherein the method or use is for improving the performance of a        subject or for improving digestibility of a raw material in a        feed (e.g. nutrient digestibility) or for improving feed        efficiency in a subject or for reducing the viscosity of the        intestinal content of a subject.    -   11. The method or use according to any one of the preceding        paragraphs wherein the xylanase comprises a polypeptide sequence        shown herein as SEQ ID No. 3, SEQ ID No. 2, SEQ ID No. 1, SEQ ID        No. 9, SEQ ID No. 10, SEQ ID No. 11 or SEQ ID No. 15; or a        variant, homologue, fragment or derivative thereof having at        least 85% identity with SEQ ID No. 3 or SEQ ID No. 2 or SEQ ID        No. 1 or SEQ ID No. 9 or SEQ ID No. 10 or SEQ ID No. 11 or SEQ        ID No. 15; or a polypeptide sequence which comprises SEQ ID No.        3, SEQ ID No. 2, SEQ ID No. 1, SEQ ID No. 9, SEQ ID No. 10, SEQ        ID No. 11, or SEQ ID No. 15 with a conservative substitution of        at least one of the amino acids; or a xylanase which is encoded        by a nucleotide sequence shown herein as SEQ ID No. 6, SEQ ID        No. 5, SEQ ID No. 4, SEQ ID No. 12, SEQ ID No. 13, SEQ ID No.        14, SEQ ID No. 16, SEQ ID No. 17 or SEQ ID No. 18, or a        nucleotide sequence which can hybridize to SEQ ID No. 6, SEQ ID        No. 5, SEQ ID No. 4, SEQ ID No. 12, SEQ ID No. 13, SEQ ID No.        14, SEQ ID No. 16, SEQ ID No. 17 or SEQ ID No. 18 under high        stringency conditions, or a nucleotide sequence which has at        least 85% identity with SEQ ID No. 6, SEQ ID No. 5, SEQ ID No.        4, SEQ ID No. 12, SEQ ID No. 13, SEQ ID No. 14, SEQ ID No. 16,        SEQ ID No. 17 or SEQ ID No. 18, or a nucleotide sequence which        differs from SEQ ID No. 6 or SEQ ID No. 5 or SEQ ID No. 4 or SEQ        ID No. 12 or SEQ ID No. 13 or SEQ ID No. 14 or SEQ ID No. 16 or        SEQ ID No. 17 or SEQ ID No. 18 due to the degeneracy of the        genetic code.    -   12. The method or use according to any one of the preceding        paragraphs wherein the xylanase comprises a polypeptide sequence        shown herein as SEQ ID No. 3, SEQ ID No. 2, SEQ ID No. 1, SEQ ID        No. 9, SEQ ID No. 10, SEQ ID No. 11 or SEQ ID No. 15; or a        variant, homologue, fragment or derivative thereof having at        least 95% identity with SEQ ID No. 3 or SEQ ID No. 2 or SEQ ID        No. 1 or SEQ ID No. 9 or SEQ ID No. 10 or SEQ ID No. 11 or SEQ        ID No. 15; or a polypeptide sequence which comprises SEQ ID No.        3, SEQ ID No. 2, SEQ ID No. 1, SEQ ID No. 9, SEQ ID No. 10, SEQ        ID No. 11 or SEQ ID No. 15 with a conservative substitution of        at least one of the amino acids; or a xylanase which is encoded        by a nucleotide sequence shown herein as SEQ ID No. 6, SEQ ID        No. 5, SEQ ID No. 4, SEQ ID No. 12, SEQ ID No. 13, SEQ ID No.        14, SEQ ID No. 16, SEQ ID No. 17 or SEQ ID No. 18, or a        nucleotide sequence which can hybridize to SEQ ID No. 6, SEQ ID        No. 5, SEQ ID No. 4, SEQ ID No. 12, SEQ ID No. 13, SEQ ID No.        14, SEQ ID No. 16, SEQ ID No. 17 or SEQ ID No. 18 under high        stringency conditions, or a nucleotide sequence which has at        least 95% identity with SEQ ID No. 6, SEQ ID No. 5, SEQ ID No.        4, SEQ ID No. 12, SEQ ID No. 13, SEQ ID No. 14, SEQ ID No. 16,        SEQ ID No. 17 or SEQ ID No. 18, or a nucleotide sequence which        differs from SEQ ID No. 6 or SEQ ID No. 5 or SEQ ID No. 4 or SEQ        ID No. 12 or SEQ ID No. 13 or SEQ ID No. 14 or SEQ ID No. 16 or        SEQ ID No. 17 or SEQ ID No. 18 due to the degeneracy of the        genetic code.    -   13. The method or use according to any one of the preceding        paragraphs wherein the xylanase comprises a polypeptide sequence        shown herein as SEQ ID No. 3, SEQ ID No. 2, SEQ ID No. 1, SEQ ID        No. 9, SEQ ID No. 10, SEQ ID No. 11 or SEQ ID No. 15; or a        variant, homologue, fragment or derivative thereof having at        least 99% identity with SEQ ID No. 3 or SEQ ID No. 2 or SEQ ID        No. 1 or SEQ ID No. 9 or SEQ ID No. 10 or SEQ ID No. 11 or SEQ        ID No. 15; or a polypeptide sequence which comprises SEQ ID No.        3, SEQ ID No. 2, SEQ ID No. 1, SEQ ID No. 9, SEQ ID No. 10, SEQ        ID No. 11 or SEQ ID No. 15 with a conservative substitution of        at least one of the amino acids; or a xylanase which is encoded        by a nucleotide sequence shown herein as SEQ ID No. 6, SEQ ID        No. 5, SEQ ID No. 4, SEQ ID No. 12, SEQ ID No. 13, SEQ ID No.        14, SEQ ID No. 16, SEQ ID No. 17 or SEQ ID No. 18, or a        nucleotide sequence which can hybridize to SEQ ID No. 6, SEQ ID        No. 5, SEQ ID No. 4, SEQ ID No. 12, SEQ ID No. 13, SEQ ID No.        14, SEQ ID No. 16, SEQ ID No. 17 or SEQ ID No. 18 under high        stringency conditions, or a nucleotide sequence which has at        least 97% (e.g. at least 97.7% or at least 98%) identity with        SEQ ID No. 6, SEQ ID No. 5, SEQ ID No. 4, SEQ ID No. 12, SEQ ID        No. 13, SEQ ID No. 14, SEQ ID No. 16, SEQ ID No. 17 or SEQ ID        No. 18, or a nucleotide sequence which differs from SEQ ID No. 6        or SEQ ID No. 5 or SEQ ID No. 4 or SEQ ID No. 12 or SEQ ID No.        13 or SEQ ID No. 14 or SEQ ID No. 16 or SEQ ID No. 17 or SEQ ID        No. 18 due to the degeneracy of the genetic code.    -   14. The method or use according to paragraph 5 wherein the feed        or feedstuff comprises or consists of corn, DDGS (such as        cDDGS), wheat, wheat bran or a combination thereof.    -   15. The method or use according to paragraph 14 wherein the feed        or feedstuff is a corn-based feedstuff.    -   16. The method or use according to any one of the preceding        paragraphs wherein the xylanase is used in combination with one        or more of the enzymes selected from the group consisting of a        protease (e.g. subtilisin (E.C. 3.4.21.62) or a bacillolysin        (E.C. 3.4.24.28) or an alkaline serine protease (E.C. 3.4.21.x)        or a keratinase (E.C. 3.4.x.x)) and/or an amylase (including        a-amylases (E.C. 3.2.1.1), G4-forming amylases (E.C. 3.2.1.60),        β-amylases (E.C. 3.2.1.2) and γ-amylases (E.C. 3.2.1.3); and/or        a protease (e.g. subtilisin (E.C. 3.4.21.62) or a bacillolysin        (E.C. 3.4.24.28) or an alkaline serine protease (E.C. 3.4.21.x)        or a keratinase (E.C. 3.4.x.x)).    -   17. The method or use according to any one of the preceding        paragraphs wherein the xylanase is used in combination with an        amylase (e.g. α-amylases (E.C. 3.2.1.1)) and a protease (e.g.        subtilisin (E.C. 3.4.21.62)).    -   18. The method or use according to any one of paragraphs 1-15        wherein the enzyme is used in combination with a β-glucanase,        e.g. an endo-1,3(4)-β-glucanases (E.C. 3.2.1.6).    -   19. The method or use according to any one of the preceding        paragraphs wherein the xylanase is contacted with a mash and/or        a wort.    -   20. The method or use according to any one of the preceding        paragraphs wherein the method comprises the steps of; (a)        preparing a mash, (b) filtering-the mash to obtain a wort        and (c) fermenting the wort to obtain a fermented beverage,        wherein said xylanase is added to: (i) the mash of step (a)        and/or (ii) the wort of step (b) and/or (iii) the wort of step        (c).    -   21. The method or use according to any one of the preceding        paragraphs wherein the xylanase comprises (or consists of) a        polypeptide sequence shown herein as SEQ ID No. 3, SEQ ID No. 2        or SEQ ID No. 1, or a variant, homologue, fragment or derivative        thereof having at least 99% identity with SEQ ID No. 3 or SEQ ID        No. 2 or SEQ ID No. 1; or a polypeptide which is encoded by a        nucleotide sequence shown herein as SEQ ID No. 6, SEQ ID No. 5        or SEQ ID No. 4, or a nucleotide sequence which has at least 97%        (e.g. at least 97.7% or at least 98%) identity with SEQ ID No.        6, SEQ ID No. 5 or SEQ ID No. 4.    -   22. A fermented beverage, e.g. beer, produced by a method        according to any one of the preceding paragraphs.    -   23. A polypeptide having xylanase activity comprising (or        consisting of) a polypeptide sequence shown herein as SEQ ID No.        3, SEQ ID No. 2 or SEQ ID No. 1, or a variant, homologue,        fragment or derivative thereof having at least 99% identity with        SEQ ID No. 3 or SEQ ID No. 2 or SEQ ID No. 1; or a polypeptide        which is encoded by a nucleotide sequence shown herein as SEQ ID        No. 6, SEQ ID No. 5 or SEQ ID No. 4, or a nucleotide sequence        which has at least 97% (e.g. at least 97.7% or at least 98%)        identity with SEQ ID No. 6, SEQ ID No. 5 or SEQ ID No. 4.    -   24. The polypeptide according to paragraph 23 wherein the        xylanase in an endo-1,4-β-d-xylanase.    -   25. The polypeptide according to paragraph 23 or paragraph 24        wherein the enzyme has an optimum temperature in the range of        50-70° C., preferably about 60° C.    -   26. A polypeptide according to any one of paragraphs 23 to 25        wherein the enzyme has a pH optimum in the range of 4.6 to 7,        preferably about 6.    -   27. A polypeptide according to any one of paragraphs 23-26        wherein the enzyme is formulated with a coating or is        encapsulated.    -   28. An isolated or recombinant nucleic acid molecule comprising        (or consisting of) a polynucleotide sequence selected from the        group consisting of:        -   a. a polynucleotide sequence which encodes a polypeptide            sequence selected from the group consisting of SEQ ID No. 3,            SEQ ID No. 2 or SEQ ID No. 1, or a variant, homologue,            fragment or derivative thereof having at least 99% identity            with SEQ ID No. 3 or SEQ ID No. 2 or SEQ ID No. 1; or        -   b. a polynucleotide sequence shown herein as SEQ ID No. 6,            SEQ ID No. 5 or SEQ ID No. 4; or a nucleotide sequence which            has at least 97% (e.g. at least 97.7% or at least 98%)            identity with SEQ ID No. 6, SEQ ID No. 5 or SEQ ID No. 4.    -   29. A vector (e.g. a plasmid) comprising a polynucleotide        sequence selected from the group consisting of:        -   a. a polynucleotide sequence which encodes a polypeptide            sequence selected from the group consisting of SEQ ID No. 3,            SEQ ID No. 2 or SEQ ID No. 1, or a variant, homologue,            fragment or derivative thereof having at least 99% identity            with SEQ ID No. 3 or SEQ ID No. 2 or SEQ ID No. 1; or        -   b. a polynucleotide sequence shown herein as SEQ ID No. 6,            SEQ ID No. 5 or SEQ ID No. 4; or a nucleotide sequence which            has at least 97% (e.g. at least 97.7% or at least 98%)            identity with SEQ ID No. 6, SEQ ID No. 5 or SEQ ID No. 4.    -   30. A host cell comprising the nucleic acid of paragraph 28 or a        vector of paragraph 29.    -   31. A method of degrading a xylan-containing material        (preferably an insoluble arabinoxylan-containing material)        comprising admixing the material with a polypeptide according to        any one of paragraphs 23-27.    -   32. A feed additive composition comprising (or consisting        essentially of or consisting of) a xylanase comprising a        polypeptide sequence shown herein as SEQ ID No. 3, SEQ ID No. 2,        SEQ ID No. 1, SEQ ID No. 9, SEQ ID No. 10, SEQ ID No. 11 or SEQ        ID No. 15; or a variant, homologue, fragment or derivative        thereof having at least 75% identity with SEQ ID No. 3 or SEQ ID        No. 2 or SEQ ID No. 1 or SEQ ID No. 9 or SEQ ID No. 10 or SEQ ID        No. 11 or SEQ ID No. 15; or a polypeptide sequence which        comprises SEQ ID No. 3, SEQ ID No. 2, SEQ ID No. 1, SEQ ID No.        9, SEQ ID No. 10, SEQ ID No. 11 or SEQ ID No. 15 with a        conservative substitution of at least one of the amino acids; or        a xylanase which is encoded by a nucleotide sequence shown        herein as SEQ ID No. 6, SEQ ID No. 5, SEQ ID No. 4, SEQ ID No.        12, SEQ ID No. 13, SEQ ID No. 14, SEQ ID No. 16, SEQ ID No. 17        or SEQ ID No. 18, or a nucleotide sequence which can hybridize        to SEQ ID No. 6, SEQ ID No. 5, SEQ ID No. 4, SEQ ID No. 12, SEQ        ID No. 13, SEQ ID No. 14, SEQ ID No. 16, SEQ ID No. 17 or SEQ ID        No. 18 under high stringency conditions, or a nucleotide        sequence which has at least 75% Identity with SEQ ID No. 6, SEQ        ID No. 5, SEQ ID No. 4, SEQ ID No. 12, SEQ ID No. 13, SEQ ID No.        14, SEQ ID No. 16, SEQ ID No. 17 or SEQ ID No. 18, or a        nucleotide sequence which differs from SEQ ID No. 6 or SEQ ID        No. 5 or SEQ ID No. 4 or SEQ ID No. 12 or SEQ ID No. 13 or SEQ        ID No. 14 or SEQ ID No. 16 or SEQ ID No. 17 or SEQ ID No. 18 due        to the degeneracy of the genetic code, or a xylanase obtainable        (or obtained) from Fusarium verticilloides or a polypeptide        according to any one of paragraphs 23 to 27.    -   33. The feed additive composition according to paragraph 32        which further comprises one or more of the enzymes selected from        the group, consisting of an amylase (including α-amylases (E.C.        3.2.1.1), G4-forming amylases (E.C. 3.2.1.60), β-amylases (E.C.        3.2.1.2) and γ-amylases (E.C. 3.2.1.3); and/or a protease (e.g.        subtilisin (E.C. 3.4.21.62) or a bacillolysin (E.C. 3.4.24.28)        or an alkaline serine protease (E.C. 3.4.21.x) or a keratinase        (E.C. 3.4.x.x)).    -   34. The feed additive composition according to paragraph 32 or        paragraph 33 which further comprises an amylase (e.g. α-amylases        (E.C. 3.2.1.1)) and a protease (e.g. subtilisin (E.C.        3.4.21.62)).    -   35. The feed additive composition according to paragraph 32        which further comprises a β-glucanase, e.g. an        endo-1,3(4)-β-glucanases (E.C. 3.2.1.6).    -   36. A premix comprising a xylanase comprising a polypeptide        sequence shown herein as SEQ ID No. 3, SEQ ID No. 2, SEQ ID No.        1, SEQ ID No. 9, SEQ ID No. 10, SEQ ID No. 11 or SEQ ID No. 15;        or a variant, homologue, fragment or derivative thereof having        at least 75% identity with SEQ ID No. 3 or SEQ ID No. 2 or SEQ        ID No. 1 or SEQ ID No. 9 or SEQ ID No. 10 or SEQ ID No. 11 or        SEQ ID No. 15; or a polypeptide sequence which comprises SEQ ID        No. 3, SEQ ID No. 2, SEQ ID No. 1, SEQ ID No. 9, SEQ ID No. 10,        SEQ ID No. 11 or SEQ ID No. 15 with a conservative substitution        of at least one of the amino acids; or a xylanase which is        encoded by a nucleotide sequence shown herein as SEQ ID No. 6,        SEQ ID No. 5, SEQ ID No. 4, SEQ ID No. 12, SEQ ID No. 13, SEQ ID        No. 14, SEQ ID No. 16, SEQ ID No. 17 or SEQ ID No. 18, or a        nucleotide sequence which can hybridize to SEQ ID No. 6, SEQ ID        No. 5, SEQ ID No. 4, SEQ ID No. 12, SEQ ID No. 13, SEQ ID No.        14, SEQ ID No. 16, SEQ ID No. 17 or SEQ ID No. 18 under high        stringency conditions, or a nucleotide sequence which has at        least 75% identity with SEQ ID No. 6, SEQ ID No. 5, SEQ ID No.        4, SEQ ID No. 12, SEQ ID No. 13, SEQ ID No. 14, SEQ ID No. 16,        SEQ ID No. 17 or SEQ ID No. 18, or a nucleotide sequence which        differs from SEQ ID No. 6 or SEQ ID No. 5 or SEQ ID No. 4 or SEQ        ID No. 12 or SEQ ID No. 13 or SEQ ID No. 14 or SEQ ID No. 16 or        SEQ ID No. 17 or SEQ ID No. 18 due to the degeneracy of the        genetic code; a polypeptide according to any one of paragraphs        23 to 27; or a feed additive composition comprising according to        any one of paragraphs 32 to 35; or a xylanase obtainable (or        obtained) from Fusarium verticilloides, and at least one mineral        and/or at least one vitamin.    -   37. The method or use according to any one of paragraphs 1-21        comprising administering a subject with a polypeptide according        to any one of paragraphs 23-27 or a feed additive composition        according to any one of paragraphs 32 to 35 or a premix        according to paragraph 36 or a xylanase obtainable (or obtained)        from Fusarium verticilloides.    -   38. A kit comprising a polypeptide according to any one of        paragraphs 23-27 or a feed additive composition according to any        one of paragraphs 32-35 or a premix according to paragraph 36        and instructions for administration.    -   39. A feedstuff comprising a feed additive composition according        to any one of paragraphs 32-35 or a feed additive composition        comprising (or consisting essentially of or consisting of) a        xylanase comprising a polypeptide sequence shown herein as SEQ        ID No. 3, SEQ ID No. 2, SEQ ID No. 1, SEQ ID No. 9, SEQ ID No.        10, SEQ ID No. 11 or SEQ ID No. 15; or a variant, homologue,        fragment or derivative thereof having at least 75% identity with        SEQ ID No. 3 or SEQ ID No. 2 or SEQ ID No. 1 or SEQ ID No. 9 or        SEQ ID No. 10 or SEQ ID No. 11 or SEQ ID No. 15; or a        polypeptide sequence which comprises SEQ ID No. 3, SEQ ID No. 2,        SEQ ID No. 1, SEQ ID No. 9, SEQ ID No. 10, SEQ ID No. 11 or SEQ        ID No. 15 with a conservative substitution of at least one of        the amino acids; or a xylanase which is encoded by a nucleotide        sequence shown herein as SEQ ID No. 6, SEQ ID No. 5, SEQ ID No.        4, SEQ ID No. 12, SEQ ID No. 13, SEQ ID No. 14, SEQ ID No. 16,        SEQ ID No. 17 or SEQ ID No. 18, or a nucleotide sequence which        can hybridize to SEQ ID No. 6, SEQ ID No. 5, SEQ ID No. 4, SEQ        ID No. 12, SEQ ID No. 13 or SEQ ID No. 14, SEQ ID No. 16, SEQ ID        No. 17 or SEQ ID No. 18 under high stringency conditions, or a        nucleotide sequence which has at least 75% identity with SEQ ID        No. 6, SEQ ID No. 5, SEQ ID No. 4, SEQ ID No. 12, SEQ ID No. 13,        SEQ ID No. 14, SEQ ID No. 16, SEQ ID No. 17 or SEQ ID No. 18, or        a nucleotide sequence which differs from SEQ ID No. 6 or SEQ ID        No. 5 or SEQ ID No. 4 or SEQ ID No. 12 or SEQ ID No. 13 or SEQ        ID No. 14 or SEQ ID No. 16 or SEQ ID No. 17 or SEQ ID No. 18 due        to the degeneracy of the genetic code; a polypeptide according        to any one of paragraphs 23-27; or a xylanase obtainable (or        obtained) from Fusarium verticilloides.    -   40. A method of preparing a feedstuff comprising admixing a feed        component with a xylanase comprising a polypeptide sequence        shown herein as SEQ ID No. 3, SEQ ID No. 2, SEQ ID No. 1, SEQ ID        No. 9, SEQ ID No. 10, SEQ ID No. 11 or SEQ ID No. 15; or a        variant, homologue, fragment or derivative thereof having at        least 75% identity with SEQ ID No. 3 or SEQ ID No. 2 or SEQ ID        No. 1 or SEQ ID No. 9 or SEQ ID No. 10 or SEQ ID No. 11 or SEQ        ID No. 15; or a polypeptide sequence which comprises SEQ ID No.        3, SEQ ID No. 2, SEQ ID No. 1, SEQ ID No. 9, SEQ ID No. 10, SEQ        ID No. 11 or SEQ ID No. 15 with a conservative substitution of        at least one of the amino acids; or a xylanase which is encoded        by a nucleotide sequence shown herein as SEQ ID No. 6, SEQ ID        No. 5, SEQ ID No. 4, SEQ ID No. 12, SEQ ID No. 13, SEQ ID No.        14, SEQ ID No. 16, SEQ ID No. 17 or SEQ ID No. 18, or a        nucleotide sequence which can hybridize to SEQ ID No. 6, SEQ ID        No. 5, SEQ ID No. 4, SEQ ID No. 12, SEQ ID No. 13, SEQ ID No.        14, SEQ ID No. 16, SEQ ID No. 17 or SEQ ID No. 18 under high        stringency conditions, or a nucleotide sequence which has at        least 75% identity with SEQ ID No. 6, SEQ ID No. 5, SEQ ID No.        4, SEQ ID No. 12, SEQ ID No. 13, SEQ ID No. 14, SEQ ID No. 16,        SEQ ID No. 17 or SEQ ID No. 18, or a nucleotide sequence which        differs from SEQ ID No. 6 or SEQ ID No. 5 or SEQ ID No. 4 or SEQ        ID No. 12 or SEQ ID No. 13 or SEQ ID No. 14 or SEQ ID No. 16 or        SEQ ID No. 17 or SEQ ID No. 18 due to the degeneracy of the        genetic code; a polypeptide according to paragraphs 23-27, or a        xylanase obtainable (or obtained) from Fusarium verticilloides        or a feed additive composition according to any one of        paragraphs 32-35.    -   41. A polypeptide, nucleic acid, vector, host cells, methods,        uses and kits as generally described herein with reference to        the Figures and Examples.

The invention will now be described, by way of example only, withreference to the following Figures and Examples.

EXAMPLES Example 1

Cloning of Fusarium verticillioides Xylanase FveXyn4)

Genomic DNA isolated from a strain of Fusarium verticillioides was usedfor amplifying a xylanase gene. The sequence of the cloned gene, calledthe FveXyn4 gene, is depicted in SEQ ID No. 4. The protein encoded bythe FveXyn4 gene is depicted in SEQ ID No. 1. The protein product ofgene FveXyn4 belongs to glycosyl hydrolase family 10 (GH10) based on thePFAM search (http://pfam.sanger.ac.uk/). At the N-terminus, FveXyn4protein has a 15 amino acid signal peptide predicted by SignalP-NN(Emanuelsson et at., Nature Protocols, 2:953-971, 2007). This indicatesthat FveXyn4 is a secreted glycosyl hydrolase.

Example 2

Expression of FveXyn4 Protein

The FveXyn4 gene was amplified from genomic DNA of Fusariumverticillioides using the following primers: Primer 15′-caccATGAAGCTGTCTTCTTTCCTCTA-3′ (SEQ ID No. 7), and Primer 25′-TTTTTAGCGGAGAGCGTTGACAACAGC-3′ (SEQ ID No. 8). The PCR product wascloned into pENTR/D-TOPO vector (Invitrogen K2400) to generate theFveXyn4 pEntry plasmid. The expression plasmid pZZH254 was obtained byGateway cloning reaction between the FveXyn4 pEntry plasmid and pTrex3gMexpression vector (described in US 2011/0136197 A1) using Gateway® LRClonase® II enzyme kit (Invitrogen 11791). A map of plasmid pZZH254 isprovided as FIG. 10. The sequence of the FveXyn4 gene was confirmed byDNA sequencing (SEQ ID No. 4). The plasmid pZZH254 was transformed intoa quad deleted Trichoderma reesei strain (described in WO 05/001036)using biolistic method (Te'o V S et al., J Microbiol Methods, 51:393-9,2002).

Following sequence confirmation, protoplasts of a quad deleted T. reeseistrain (described in WO 05/001036) were transformed with the expressionplasmid pTTT-Ate CA1 using the PEG protoplast method (Penttila et al,Gene, 61:155-164, 1987). For protoplast preparation, spores were grownfor about 10 hours at 24° C. in Trichoderma Minimal Medium MM (20 g/Lglucose, 15 g/L KH₂PO₄, pH 4.5, 5 g/L (NH₄)2SO₄, 0.6 g/L MgSO₄x7H₂O, 0.6g/L CaCl₂x2H₂O, 1 ml of 1000×T. reesei Trace elements solution (175 g/LCitric Acid anhydrous, 200 g/L FeSO₄x7H₂O, 16 g/L ZnSO₄x7H₂O, 3.2 g/LCuSO₄, 1.4 g/L MnSO₄xH2O, and 0.8 g/L Boric Acid). Germinating sporeswere harvested by centrifugation and treated with 30 mg/mL Vinoflow FCE(Novozymes, AG Switzerland) solution for from 7 hours to overnight at30° C. at 100 rpm to lyse the fungal cell walls. Protoplasts were washedin 0.1 M Tris HCl buffer (pH 7) containing 0.6 M sorbitol andresuspended in 10 mM Tris HCl buffer (pH 7.5) containing 1.2 M sorbitoland 10 mM calcium chloride. For PEG transformation, approximately 1 μgof DNA and 1-5×10⁷ protoplasts in a total volume of 200 μl were treatedwith 2 ml of 25% PEG solution, diluted with 2 volumes of 1.2 Msorbitol/10 mM Tris, pH 7.5/10 mM CaCl₂ solution. Transformants wereselected on a medium containing acetamide as a sole source of nitrogen(acetamide 0.6 g/L; cesium chloride 1.68 g/L; glucose 20 g/L; potassiumdihydrogen phosphate 15 g/L; magnesium sulfate heptahydrate 0.6 g/L;calcium chloride dihydrate 0.6 g/L; iron (II) sulfate 5 mg/L; zincsulfate 1.4 mg/L; cobalt (II) chloride 1 mg/L; manganese (II) sulfate1.6 mg/L; agar 20 g/L; pH 4.25). Transformed colonies (about 50-100)appeared in about 1 week. After growth on acetamide plates, the sporeswere collected and reselected on acetamide plates. After 5 days, thespores were collected using 10% glycerol, and 1×10⁸ spores wereinoculated in a 250 ml shake flask with 30 ml Glucose/Sophorose definedmedium for protein expression. Protein expression was confirmed bySDS-PAGE. The spore suspension was subsequently grown in a 7 L fermentorin a defined medium containing 60% glucose-sophorose feed.Glucose/Sophorose defined medium (per liter) consists of (NH₄)2SO₄ 5 g,PIPPS buffer 33 g, Casamino Acids 9 g, KH₂PO₄ 4.5 g, CaCl₂ (anhydrous) 1g, MgSO₄.7H₂O 1 g, pH to 5.5 adjusted with 50% NaOH with Milii-Q H₂O tobring to 966.5 mL. After sterilization, the following were added: 26 mL60% Glucose/Sophrose, and 400×T. reesei Trace Metals 2.5 mL.

FveXyn4 was purified from concentrated fermentation broth of a 7 Lfermentor culture using two chromatography columns. Concentratedfermentation broth buffered in 20 mM sodium phosphate buffer pH 6.0containing 1 M ammonium sulfate was loaded on a hydrophobic interactionchromatography column (Sepharose Phenyl FF, 26/10). The protein waseluted from the column using a linear gradient of equilibration/washbuffer to 20 mM sodium phosphate buffer pH 6.0. The fraction containingFveXyn4 protein was loaded onto a gel filtration column (HiLoad Superdex75 pg 26/60), and the mobile phase used was 20 mM sodium phosphate, pH7.0, containing 0.15 M NaCl. The purified protein was concentrated usinga 3K Amicon Ultra-15 device and the concentrated protein fraction wasused in further studies.

The nucleotide sequence of FveXyn4 gene from expression plasmid pZZH254is set forth as SEQ ID No. 4. The signal sequence is shown in bold(upper case), and the predicted intron is shown in bold and lowercase.

The amino acid sequence of FveXyn4 protein expressed from plasmidpZZH254 is set forth as SEQ ID No. 1. The signal sequence predicted bySignalP-NN software is shown underlined. This is the pre-pro-protein.

The amino acid sequence of the predicted mature form of FveXyn4 proteinis set forth as SEQ ID No. 3. This is the active form of the enzyme. SEQID No. 2 shows the pro-protein, i.e. before post-translationalmodification. Depending on the host the post-translation modificationmay vary and therefore the present invention also encompasses mature,active forms of SEQ ID No. 2.

Example 3

Xylanase Activity of FveXyn4

FveXyn4 belongs to the glycosyl hydrolase 10 family (GH10, CAZy number).The beta 1-4 xylanase activity of FveXyn4 was measured using 1% xylanfrom birch wood (Sigma 95588) or 1% arabinoxylan from wheat flour(Megazyme P-WAXYM) as substrates. The assay was performed in 50 mMsodium citrate pH 5.3, 0.005% Tween-80 buffer at 50° C. for 10 minutes.

The released reducing sugar was quantified by reaction with 3,5-Dinitrosalicylic acid and measurement of absorbance at 540 nm. Theenzyme activity is quantified relative to a xylose standard curve. Inthis assay, one xylanase unit (U) is defined as the amount of enzymerequired to generate 1 micromole of xylose reducing sugar equivalentsper minute under the conditions of the assay.

Example 4

pH Profile of FveXyn4

The pH profile of FveXyn4 was determined using xylan from birch wood(Sigma 95588) as substrate. The assay was performed in SodiumCitrate/Sodium Phosphate buffer solution adjusted to pH values between 2and 9. Birchwood xylan (2% solution) dissolved in water was mixed withsame volume of 50 mM Citrate/Phosphate buffer solution in a 96-wellplate, and the substrate was equilibrated at 50° C. before addingenzyme. After 10 minutes, the enzyme reaction was stopped bytransferring 60 microliters of reaction mixture to a 96-well PCR platecontaining 100 microliters of DNS solution. The PCR plate was heated at95° C. for 5 minutes in a Bio-Rad DNA Engine. Then plate was cooled toroom temperature and 100 microliters were transferred from each well toa new 96-well plate. Release of reducing sugars from the substrate wasquantified by measuring the optical density at 540 nm in aspectrophotometer. Enzyme activity at each pH was reported as relativeactivity where the activity at the pH optimum was set to 100%. The pHprofile of FveXyn4 is shown in FIG. 11. FveXyn4 was found to have anoptimum pH at about 6, and was found to retain greater than 70% ofmaximum activity between pH 4.6 and 7.

Example 5

Temperature Profile of FveXyn4

The temperature optimum of purified FveXyn4 was determined by assayingfor xylanase activity at temperatures varying between 40° C. and 75° C.for 10 minutes in 50 mM sodium citrate buffer at pH 5.3. The activitywas reported as relative activity where the activity at the temperatureoptimum was set to 100%. The temperature profile of FveXyn4 is shown inFIG. 12. FveXyn4 was found to have an optimum temperature of 60° C., andwas found to retain greater than 70% of maximum activity between 45° C.and 64° C.

Example 6

Pentosan Solubilisation (Breakdown or Solubilisation of InsolubleArabinoxylan (AXinsol))

The new xylanase (FveXyn4) was cloned, expressed, purified andcharacterized and tested against a benchmark xylanase product(Econase®XT).

The ability to solubilize insoluble arabinoxylan from 4 substrates,namely wheat, wheat bran, corn and corn DDGS was used as the keyselection criteria.

The new xylanase showed strong performance on pentosan solubilisationfrom relevant feed raw materials (wheat, wheat bran, corn, corn DDGS).Surprisingly the new xylanase was far better than the benchmarks (e.g.the commercially available xylanase products) on a greater number ofsubstrates.

In particular the enzyme was surprisingly far superior to the benchmarkon the corn based materials.

The new xylanase also shows numerous advantageous properties for itsindustrial use including unexpectedly high pentosan solubilisation,particularly in feedstuffs comprising corn.

6.1 Materials and Methods

Enzyme Samples

The xylanases used in this study are:

A new GH10 xylanase from Fusarium verticilloides (designated FveXyn4)expressed in Trichoderma reesei, wherein the xylanase was used inpurified form—this enzyme may be referred to herein as FveXyn4, and thefollowing benchmark, commercially available xylanase: Econase® XT. Thisbenchmark enzyme was extracted from commercial dry formulated samples.The xylanase component from Econase® XT commercial dry formulatedsamples was extracted in a 33% (w/w) slurry using Mcllvain buffer, pH5.0. The extract was cleared using centrifugation (3000 RCF for 10 min)and filtered using a PALL Acrodisc PF syringe filter (0.8/0.2 μm Supormembrane) and subsequently heated 20 min at 70° C. After removable ofprecipitation by centrifugation (38 724 RCF for 15 min) the buffer wasreplaced by passage through a Sephadex G25 column (PD10 from Pharmacia)equilibrated with 20 mM Na Citrate, 20 mM NaCl, pH 3.4. Purification ofthe xylanase component was performed using Source 15S resin, followed byelution with a linear increasing salt gradient (NaCl in 20 mM Na Citratebuffer pH 3.4).

Econase XT® is an endo-1,4-β-xylanase (EC 3.2.1.8) produced by thestrain Trichoderma reesei RF5427 (CBS 114044), available from ABVista.

Protein concentration was determined by measuring absorption at 280 nm.The extinction coefficients were estimates from the amino acidsequences. For Econase XT the absorption at 280 nm of 1 mg/ml wascalculated to be 2.84 AU.

Feed Raw Materials

The feed used in these experiments is raw material. The feeds are eithercorn, corn DDGS, wheat or wheat bran.

Pentosan Solubilisation (AXinsol Solubilisation)

The method used for pentosan solubilisation was: 100 mg of feed rawmaterial was transferred to a 2 ml Eppendorf centrifuge tube and theprecise weight recorded. 750 μL incubation buffer (200 mM HEPES, 100 mMNaCl, 2 mM CaCl, pH 6.0) and 900 μl chloramphenicol solution (40 μg/mlin incubation buffer) was added. Enzyme of choice was added to make atotal volume of 1.8 mL.

Each sample was assayed in doublets and in parallel with a blank(incubation without exogenously added enzyme). The samples wereincubated on an Eppendorf thermomixer at 40° C. with shaking. After 2 or18 hours of incubation the supernatant was filtered using 96 wellsfilterplates (Pall Corporation, AcroPrep 96 Filter Plate, 1.0 μm Glass,NTRL, 1 mL well). After filtration the samples were stored at 4° C.until analysis for total amount of C5 sugars, arabinose and xylose.

Quantification of C5 Sugars (Pentosans)

The total amount of pentoses brought into solution was measured usingthe method of Rouau and Surget (1994, A rapid semi-automated method ofthe determination of total and water-extractable pentosan in wheatflours. Carbohydrate Polymers, 24, 123-32) with a continuous flowinjection apparatus (FIG. 7). The supernatants were treated with acid tohydrolyse polysaccharides to monosugars. Phloroglucinol (1, 3,5-trihydroxybenzen) was added for reaction with monopentoses andmonohexoses, which forms a coloured complex. By measuring the differencein absorbance at 550 nm compared to 510 nm, the amount of pentoses inthe solution was calculated using a standard curve. Unlike thepentose-phloroglucinol complex, the absorbance of thehexose-phloroglucinol complex is constant at these wavelengths. Glucosewas added to the phloroglucinol solution to create a constant glucosesignal and further ensure no interference from hexose sugars.

6.2 Results and Discussion

Pentosan solubilisation was monitored in a dose response setup usingfibrous by-products of wheat (namely wheat bran) and a fibrousby-product of corn (namely cDDGS).

The results from benchmark Econase® XT and of the novel xylanase(FveXyn4) are shown in FIG. 8 (in wheat bran) and FIG. 9 (in corn DDGS).

FIG. 8 shows pentosan (C-5 sugar) release (solubilisation of pentosans)from wheat bran as a function of xylanase dosage. The xylanases usedwere the xylanase of the present invention (FveXyn4) compared with thebenchmark xylanase namely Econase® XT.

FIG. 9 shows solubilisation of pentosans from cDDGS as a function ofxylanase dosage. The xylanases used were the xylanase of the presentinvention (FveXyn4) compared with the benchmark xylanase namely Econase®XT.

Econase® XT performs well on wheat but shows no or limited effect oncorn.

This indicates a clear difference in substrate specificity compared tofor instance FveXyn4, surprisingly FveXyn4 is good at breaking downAXinsol (e.g. solubilising pentosans) in both wheat and corn basedsubstrates. Typically xylanase enzymes are poor performers on corn basedproducts. Surprisingly the present enzyme is capable of dissolvinginsoluble arabinoxylans (AXinsol) in both wheat and corn basedsubstrates.

It is worth noting that the tested commercially available xylanase(Econase® XT) did not show significant solubilisation of corn. In fact,there are very few commercially available xylanases that showsignificant ability to dissolve AXinsol in corn (or solubilisation ofcorn). This is where the present enzyme differs significantly.

Example 7

Viscosity Reduction in In Vitro Animal Model Assay

Viscosity reduction on wheat was determined using a modified version ofthe procedure described by Bedford & Classen (1993 Poultry Sci., 72,137-143). 3.6 mL of pepsin solution (2000 U/mL in 0.1 N HCl) was mixedwith 2.4 g wheat prior to addition of the indicated amount of xylanase(FveXyn4) followed by 45 min incubation at 40° C. 1.2 ml pancreatinsolution (8 mg/mL in 1 M MES, pH 6.8) was then mixed into the slurryresulting in a final pH at 6.0. The sample was allowed to incubate for60 min at 40° C. with mixing after 30 and 60 min. The sample was thenplaced on ice for 5 min to stop the reaction and centrifuged 10 min at3320 RCF followed by filtration through a 0.45 μm filter to obtain aclear supernatant. Sample viscosity was then measured at 20° C. using aBrookfield digital viscometer (model DV-I+, Brookfield EngineeringLaboratories, Stoughton, Mass. 02172, USA) fitted with a CPE-40 cone andplate. Each data point is the average of three repetitions.

The results are shown in FIG. 13. As can be seen even under theseconditions (which conditions attempt to mimic the environment in thesmall intestine of an animal) FveXyn4 reduces the viscosity.

Example 8

Viscosity Reduction in Grain-Based Material (e.g. For BiofuelProduction)

In the European fuel alcohol industry, small grains like wheat, barleyand rye are common raw materials, in contrast to the US where mainlycorn is used. These small grains contain, next to starch, high levels ofnon-starch polysaccharide polymers (NSP), like cellulose, beta-glucanand hemicellulose.

The ratio in which the different NSPs are represented differ for eachfeedstock.

NSPs give high viscosity to grain mashes due to their largewater-binding capacity. High viscosity has a negative impact on ethanolproduction since it will limit the solid concentration that can be usedin mashing and it will reduce the energy efficiency of the process. Inaddition, residual hemicelluloses present throughout the process maycontribute to fouling in heat exchangers and distillation equipment. Thelargest impact of a high viscosity is seen when a mash is cooled tofermentation temperature (32° C.). This explains that the viscosityneeds to be reduced in the process anywhere before the cooling step.

8.1 Materials and Methods

A Rapid Visco Analyzer (RVA 4500) from Perten Instruments was used tomeasure viscosity profiles of a wheat mash. The RVA 4500 is a cookingstirring viscometer with ramped temperature and variable shear that canbe used to determine the quality and processing characteristics ofstarch in grains, tubers, flours and extruded and cooked foods andfeeds. There are also applications for protein foods, ingredients suchas modified starches and hydrocolloids and malting and brewing.

The wheat mash (50 grams of a 30% DS, 34.86% ‘as is’ slurry) wasprepared according to the following protocol:

-   Weigh 17.42 grams of wheat-   In a 50 ml beaker glass, weigh 32.58 grams of tap water and add 114    μl 4N H₂SO₄-   Add the wheat to the water and stir for 5 minutes at maximum speed    (approx. 500 rpm) with an overhead stirrer-   Transfer 25.0 grams of this slurry (pH 5.2) to an RVA cup, add    50-fold diluted enzymes and start RVA run-   Divide all slurry over two 15-ml Greiner centrifuge tubes and    centrifuge 10 minutes at 3500 rpm (2547×g)-   Determine layer separation (read volume scale on centrifuge tube)

The term “dry solids content (DS)” refers to the total solids (dissolvedand undissolved) of a slurry (in %) on a dry weight basis. At the onset,“initial DS” refers to the dry solids in the slurry at time zero. As thehydrolysis reaction proceeds, the portion of DS that are dissolved canbe referred to as “Syrup DS” as well as “Supernatant DS”.

In each experiment (25 grams of slurry), xylanases FveXyn4 and FoxXyn2were dosed at 25 μg protein (per 8.71 g wheat ‘as is’), corresponding to2.9 μg protein per g wheat ‘as is’. XYLATHIN™ was dosed at 4 μg proteinper g wheat ‘as is’. SPEZYME® CL was dosed at 2.0 AAU/g DS (2.3 AAU/g‘as is’).

A standard wheat liquefaction was mimicked in the RVA. Pretreatment wasperformed for 20 minutes at 60° C., followed by a liquefaction step for30 minutes at 85° C. After pretreatment and liquefaction, slurry wascooled down to 32° C., to determine viscosity at fermentationconditions. Tables below contain the viscosities after each step,complete RVA profiles are shown in FIGS. 14 and 18.

In one experiment, the performance of FveXyn4, Fusarium verticilloideswas compared to Verenium's Xylathin™ (benchmark). Enzyme in Xylathin™ isa glycosyl hydrolase family 11 from uncultured (metagenomic) bacterium(U.S. Pat. No. 7,504,120).

TABLE 8.1 Viscosity (mPa*s) Xylathin ™, Blank benchmark FveXyn4 Afterpretreatment 626 361 183 (1200 sec. process time) After liquefaction 495177 106 (3120 sec. process time) At fermentation 1005 379 222temperature (3660 sec. process time) Brix after RVA run 27.50 ± 0.2727.89 28.18

The Brix value is a measure of the concentration of dissolved substance,for instance sugars, in a watery liquid, or syrup in case of grainprocessing. It is used to quantify the solubilization/degradationprocess of the sugars. The higher the values, the more sugars aredissolved. Reference values are needed for comparison. Brix values areexpressed in degrees and are measured using a refractometer.

These data together with FIG. 14 show that FveXyn4 outperforms Xylathin™on viscosity.

In another experiment, the performance of FoxXyn2, Fusarium oxysporum)was compared to Verenium's Xylathin™ (benchmark). The enzyme inXylathin™ is a glycosyl hydrolase family 11 from uncultured(metagenomic) bacterium (U.S. Pat. No. 7,504,120).

TABLE 8.2 Viscosity (mPa*s) Xylathin ™, Blank benchmark FoxXyn2 Afterpretreatment 626 361 204 (1200 sec. process time) After liquefaction 495177 115 (3120 sec. process time) At fermentation 1005 379 237temperature (3660 sec. process time) Brix after RVA run 27.50 ± 0.2727.89 30.20

These data together with FIG. 18 show that FoxXyn2 outperforms Xylathin™on viscosity.

Example 9

Wheat Gluten-Starch Separation

Separation of wheat flour into starch and gluten fractions isindustrially applied on large scale to obtain high quality A-starch andbyproducts B-starch and vital gluten.

Separation is improved by addition of xylanases.

9.1 Materials and Methods

The following assay simulates the wheat starch separation of a batterprocess at 40° C. In this assay, industrial wheat flour (Cargill) wasadded to preheated tap water (50° C.) to create a 35%DS slurry by mixing1 minute in a kitchen blender (Braun). pH of the slurry remained ‘as is’at ˜6.1. 100 gram of this slurry was transferred to the Haake VT550viscometer, which was calibrated at 40° C. After 1 minute of incubation,the enzyme solution was added to the slurry. Meanwhile, the viscosityprofile was monitored before and after enzyme addition for 15 minutes intotal. After incubation, triplicate samples of the incubated slurry andone sample of t₀ slurry were taken for a spin test. Each spin testsample has a total weight of 22.5 g, which contains 15.8-15.9 g slurrysample added to 6.6-6.7 g of disposable centrifuge tube (15 ml). Allsamples were centrifuged in a Hermle Z400 centrifuge for 15 minutes at3500 rpm. Brix values were determined from the syrup of the centrifugedsamples.

Example 9A

In one experiment, the performance of xylanase FveXyn4 in accordancewith the present invention in a wheat gluten-starch separation processwas compared to the commercial enzymes, Shearzyme Plus™ (Novozymes). Thecommercial enzyme Shearzyme Plus™ was dosed at 0.20 kg/MT DS. XylanaseFveXyn4 was tested at a dose of 1.19 g protein/MT DS.

A 35% dry solids batter (pH ‘as is’ at ˜6.1) was prepared by addingCargill wheat flour to demineralized water preheated to 50° C., whilecontinuously mixing it in a Braun blender for 1 minute at 18900 r.p.m.From this batter, 100 g was transferred to the Haake VT550 viscometer,preheated to 40° C. and stirring at v=50 1/s. The viscosity was measuredfor 1 minute, after which enzymes were added and the viscosity wasmonitored for in total 15 minutes, see results in Table 1. Theunderlined values are viscosity values lower, or in other words betterin performance, than those of the benchmark, Shearzyme Plus™. It Isclear that xylanase FveXyn4 is significantly performing better than thebenchmark Shearzyme Plus™.

In succession, three samples of the batter were immediately afterincubation put in 15 ml disposable centrifuge tubes, each with a totalweight of 22.5 g. The tubes were centrifuged for 15 minutes at 3500r.p.m. in a Hermle Z400 centrifuge, and the individual layers weremeasured and averaged for the triplicates. Table 2 shows the percentageof each layer for the blanc, a batter without enzyme addition incubatedin the Haake VT550 viscometer under the same conditions, and for all thexylanases tested. The underlined values of supernatant layer (%) areconsiderably larger than those of the benchmark, Shearzyme Plus™.

TABLE 9.1 The effect of xylanases on wheat flour viscosity in time,during a 15 minute incubation at 40° C. Viscosity in time (mPA*s)Shearzyme Time Plus ™ (min) Blanc (Benchmark) FveXyn4 0 481.13 513.00494.1 5 393.50 295.90 204.1 10 375.83 238.70 155.5 15 354.97 210.60130.7 Brix at 5.88 6.07   6.32 t₁₅

TABLE 9.2 The effect of xylanases on wheat starch separation after 15minute incubation at 40° C. Average of Shearzyme Separated Plus ™ layers(%) Blanc (Benchmark) FveXyn4 Starch 33.74 34.6 34.3 Fiber 16.84 14.013.2 Sludge 24.22 23.3 20.2 Supernatant 25.20 28.1 32.3

Example 9B

In a second experiment, the performance of xylanase FoxXyn2 in a wheatstarch separation process was compared to the commercial enzyme,Shearzyme Plus™. The commercial enzyme Shearzyme Plus™ was dosed at 0.20kg/MT DS. Xylanase FoxXyn2 was tested at a dose of 1.19 gr protein/MTDS.

A 35% dry solids batter (pH ‘as is’ at ˜6.1) was prepared by addingCargill wheat flour to demineralized water preheated to 50° C., whilecontinuously mixing it in a Braun blender for 1 minute at 18900 r.p.m.From this batter, 100 gr. was transferred to the Haake VT550 viscometer,preheated to 40° C. and stirring at v=50 1/s. The viscosity was measuredfor 1 minute, after which enzymes were added and the viscosity wasmonitored for in total 15 minutes, see results in Table 1. Theunderlined values are viscosity values lower, or in other words betterin performance, than those of the benchmark, Shearzyme Plus™. It isclear that xylanase FoxXyn2 is significantly performing better than thebenchmark Shearzyme Plus™.

In succession, three samples of the batter were immediately afterincubation put in 15 ml disposable centrifuge tubes, each with a totalweight of 22.5 gr. The tubes were centrifuged for 15 minutes at 3500r.p.m. in a Hermle Z400 centrifuge, and the individual layers weremeasured and averaged for the triplicates. Table 2 shows the percentageof each layer for the blanc, a batter without enzyme addition incubatedin the Haake VT550 viscometer under the same conditions, and for all thexylanases tested. The underlined values of supernatant layer (%) areconsiderably larger than those of the benchmark, Shearzyme Plus.

TABLE 9.3 The effect of xylanases on wheat flour viscosity in time,during a 15 minute incubation at 40° C. Viscosity in time (mPA*s)Shearzyme Time Plus (min) Blanc (Benchmark) FoxXyn2 0 481.13 513.00 488.4 5 393.50 295.90 203.05 10 375.83 238.70 152.25 15 354.97 210.60126.35 Brix 5.88 6.07  6.35 at t₁₅

TABLE 9.4 The effect of xylanases on wheat starch separation after 15minute incubation at 40° C. Average of Shearzyme Separated Plus layers(%) Banc (Benchmark) FoxXyn2 Starch 33.74 34.6 34.1 Fiber 16.84 14.013.5 Sludge 24.22 23.3 19.9 Supernatant 25.20 28.1 32.5

Example 10

Cloning of Fusarium oxysporum Xylanase FoxXyn2

The nucleotide sequence of the FoxXyn2 gene isolated from Fusariumoxysporum is set forth as SEQ ID Nos 12, 13 and 14. The predicted intronis shown in SEQ ID No. 12 (FIG. 19) in italics and lowercase.

The amino acid sequence of the FoxXyn2 precursor protein is set forth asSEQ ID No. 9 (FIG. 15). The predicted signal sequence is shown initalics and lowercase.

The amino acid sequence of the predicted mature forms of FoxXyn2 is setforth as SEQ ID Nos. 10 and 11 (shown in FIGS. 15 and 17). SEQ ID No. 10shows a section of the polypeptide that may be cleaved before fullmaturation of the protein. The active form of the protein may be with orwithout this section, and thus the active protein may have SEQ ID No. 10or SEQ ID No. 11.

The protein product of gene FoxXyn2 belongs to glycosyl hydrolase family10. This suggests that FoxXyn2 is a secreted glycosyl hydrolase.

Example 11

Expression of FoxXyn2 Protein

The FoxXyn2 gene was amplified from genomic DNA of Fusarium oxysporumusing the following primers: Primer 15′-ccgcggccgcaccATGAAGCTGTCTTCCTTCCTCTACACC-3′ (SEQ ID NO:19), andPrimer 2 5′-ccggcgcgcccttaTTAGCGGAGAGCGTTGACAACAG-3′ (SEQ ID NO:20).After digested with Not I and Asc I, the PCR product was cloned intopTrex3gM expression vector (described in US 2011/0136197 A1) digestedwith the same restriction enzymes, and the resulting plasmid was labeledpZZH135. A plasmid map of pZZH135 is provided in FIG. 22. the sequenceof the FoxXyn2 gene was confirmed by DNA sequencing.

The plasmid pZZH135 was transformed into a quad deleted Trichodermareesei strain (described in WO 05/001036, incorporated herein byreference) using biolistic method (taught in Te'o V S et al., JMicrobiol Methods, 51:393-9, 2002). The protein isolated from theculture supernatant after filtration was used to perform SDS-PAGEanalysis and xylanase activity assay to confirm enzyme expression.

The nucleotide sequence of FoxXyn2 gene from expression plasmid pZZH135is set forth as SEQ ID No. 12 (FIG. 19). The signal sequencers shown inbold, and the predicted intron is shown in italics and lowercase.

The amino acid sequence of FoxXyn2 protein expressed from plasmidpZZH135 is set forth as SEQ ID No. 9 (FIG. 15). The signal sequence isshown in italics.

The amino acid sequence of the mature form of FoxXyn2 protein is setforth as SEQ ID No. 10 (FIG. 16).

FoxXyn2 protein was purified from culture supernatant using affinitychromatography resin Blue Sepharose, 6FF, and samples were used forbiochemical characterization as described in subsequent examples.

Example 12

Xylanase Activity of FoxXyn2

FoxXyn2 belongs to the glycosyl hydrolase 10 family (GH10, CAZy number).The beta 1-4 xylanase activity of FoxXyn2 was measured using 1% xylanfrom birch wood (Sigma 95588) or 1% arabinoxylan from wheat flour(Megazyme P-WAXYM) as substrates. The assay was performed in 50 mMsodium citrate pH 5.3, 0.005% Tween-80 buffer at 50° C. for 10 minutes.

The released reducing sugar was quantified by reaction with 3,5-Dinitrosalicylic acid and measurement of absorbance at 540 nm. Theenzyme activity is quantified relative to a xylose standard curve. Inthis assay, one xylanase unit (U) is defined as the amount of enzymerequired to generate 1 micromole of xylose reducing sugar equivalentsper minute under the conditions of the assay.

Example 13

pH Profile of FoxXyn2

The pH profile of FoxXyn2 was determined using xylan from birch wood(Sigma 95588) as substrate. The assay was performed in SodiumCitrate/Sodium Phosphate buffer solution adjusted to pH values between 2and 9. Birchwood xylan (2% solution) dissolved in water was mixed withequal volume of 50 mM Citrate/Phosphate buffer solution in a 96-wellplate, and the substrate was equilibrated at 50° C. before addingenzyme. After 10 minutes, the enzyme reaction was stopped bytransferring 60 microliters of reaction mixture to a 96-well PCR platecontaining 100 microliters of DNS solution. The PCR plate was heated at95° C. for 5 minutes in a Bio-Rad DNA Engine. Then plate was cooled toroom temperature and 100 microliters were transferred from each well toa new 96-well plate. Release of reducing sugars from the substrate wasquantified by measuring the optical density at 540 nm in aspectrophotometer. Enzyme activity at each pH was reported as relativeactivity where the activity at the pH optimum was set to 100%. The pHprofile of FoxXyn2 is shown in FIG. 23. FoxXyn2 was found to have anoptimum pH at about 6, and was found to retain greater than 50% ofmaximum activity between pH 4.5 and 6.5.

Example 14

Temperature Profile of FoxXyn2

The temperature optimum of purified FoxXyn2 was determined by assayingfor xylanase activity at temperatures varying between 45° C. and 94° C.for 10 minutes in 50 mM sodium citrate buffer at pH 5.3. The activitywas reported as relative activity where the activity at the temperatureoptimum was set to 100%. The temperature profile of FoxXyn2 is shown inFIG. 24. FoxXyn2 was found to have an optimum temperature of 60° C., andwas found to retain greater than 50% of maximum activity between 40° C.and 65° C.

Example 15

FveXyn4 in Animal Feed—Pigs

15.1 Materials and Methods

Two experiments are conducted to evaluate the efficacy of FveXyn4 incorn/corn DDGS based diets (Experiment 1) and wheat/wheat bran baseddiets (Experiment 2). An Animal Care and Use Committee approves the useof the pigs and relevant welfare guidelines for the Country are used.Each experiment used a total of 48 pigs ([♀Yorkshire×Landrace]×♂Duroc)housed in groups of two and each pen contained a gilt and a barrow. Eachpen has a smooth transparent plastic sides and plastic-covered expandedmetal sheet flooring in a temperature-controlled room (22±2° C.).

TABLE 15.1 Basal diets Item Experiment 1 Experiment 2 Corn 41.44 US cornDDGS 40.00 Hard Wheat 56.57 Wheat Bran 5.06 Wheat middlings 19.94Soybean Meal 15.00 13.50 Tallow 0.99 L-Lysine HCl 0.75 0.73DL-Methionine 0.10 0.17 L-Threonine 0.25 0.30 L-Tryptophan 0.06 0.01Digestibility marker (celite) 0.30 0.30 Salt 0.30 0.47 Limestone 1.301.38 Monocalcium Phosphate 0.00 0.08 Vitamins/Trace minerals premix¹0.50 0.50 Calculated provisions Crude protein, % 21.44 19.11 Net energy,MJ/kg 8.88 8.88 SID Lysine g/NE MJ 1.31 1.31 SID Lysine, % 1.16 1.16 SIDMethionine, % 0.35 0.35 Neutral detergent fibre, % 20.63 17.93 Calcium,% 0.67 0.68 Available phosphorous, % 0.22 0.22 ¹The vitamin and tracemineral premix provided the following (per kg of diet): vitamin A,11,000 IU; vitamin D3, 2,756 IU; vitamin E, 55 IU; vitamin B12, 55 μg,riboflavin, 16,000 mg; pantothenic acid, 44.1 mg; niacin, 82.7 mg; Zn,150 mg; Fe, 175 mg; Mn, 60 mg; Cu, 17.5 mg; I, 2 rng; and Se, 0.3 mg

Respective basal diets are formulated to meet the NRC nutrientsrecommendations for swine (NRC, 1998 Table 15.1). In each experiment,one batch of the basal diet is manufactured and split into four portionsand each portion subsequently mixed with additives identified in Table15.2. In experiment 1, pigs are offered the experimental diets for 42days, whilst in experiment 2 pigs are offered the experimental diets offor 21 days. Feed and water is freely available at all times duringexperimentation. There are 4 replicate pens per treatment in eachexperiment; pen allocation to the treatments is randomized based on pigbody weight at the start of the experiment. Body weight and Feed intakeare recorded on a weekly basis and used to calculate feed conversionratio. The growth performance data (BW, ADFI, ADG and FCR) are subjectedto mixed-liner model using the GLM procedures of SAS.

TABLE 15.2 Treatments identification Phytase¹ Diet Treatment ID (FTU/kgof feed) Xylanase Control 1 500 FTU 0 Control + FveXyn4 2 500 FTU 2000U/kg Control + Commercial 3 500 FTU 75 ppm xylanase² ¹Phytase fromDanisco Animal Nutrition ²Econase XT ® from AB Vista (sometimes referredto herein as AB Vista)

15.2 Results and Discussion

Compared with the control, FveXyn4 improves animal weight gain in bothcorn (Table 15.3) and wheat (Table 15.4) based diets. Furthermore,FveXyn4 has numerically superior effects on growth performance relativeto commercially available feed xylanase, particularly in corn baseddiets. This demonstrates the efficaciousness of FveXyn4 in mitigatingthe negative effects of the insoluble and soluble fibrous fractions incereal ingredients on swine performance and shows better utilization ofenergy contained in the fibre component of cereal grains.

TABLE 15.3 Experiment 1: Effect of new xylanase on growth performance ofgrowing pigs fed corn-corn DDGS based diets Initial Final Average bodybody daily Average Feed weight, weight, feed intake, daily gain,conversion kg kg grams/day grams/day ratio, g/g Control 32.5 68.18b 2285849b 2.69 FveXyn4 32.9 74.09a 2391 972a 2.46 Commercial 32.8 71.0ab 2220909ab 2.43 xylanase (Econase ® XT) SEM 0.69 2.18 154 43.5 0.11

TABLE 15.4 Experiment 2: Effect of new xylanase on growth performance ofgrowing pigs fed wheat-wheat bran based diets Initial Final Average bodybody daily Average Feed weight, weight, feed intake, daily gain,conversion kg kg grams/day grams/day ratio, g/g Control 50.5 66.3 2221753b 2.98a FveXyn4 49.0 69.1 2332 955a 2.46ab Commercial 49.7 69.4 2464935a 2.66ab xylanase (Econase ® XT) SEM 1.53 1.99 172 56.4 0.24

Example 16

FveXyn4 in Animal Feed—Pigs

16.1 Materials and Methods

An experiment is conducted to evaluate the efficacy of FveXyn4 inwheat/wheat bran based diets. Animal use and experimental procedures areapproved by an Animal Care Committee and relevant welfare guidelines forthe Country were used. Growing gilts ([Yorkshire×Landrace♀]×Duroc♂) areobtained from a Research Unit and upon arrival, pigs are weighed andbased on body weight randomly assigned to pens to give 8 pens pertreatment. The pens are equipped with a feeder, a nipple type drinkerand plastic-covered expanded metal floors and a wall partitioningbetween pens that allowed visual contact with pigs in adjacent pens. Theroom temperature was maintained at 22±2° C. throughout the experiment.

TABLE 16.1 Composition of the basal diet Item Level Hard Wheat 56.57Wheat Bran 5.06 Wheat middlings 19.94 Soybean Meal 13.50 Soybean oil0.99 L-Lysine HCl 0.73 DL-Methionine 0.17 L-Threonine 0.30 L-Tryptophan0.01 Digestibility marker (celite) 0.30 Salt 0.47 Limestone 1.38Monocalcium phosphate 0.08 Vitamins/Trace minerals premix¹ 0.50Calculated provisions Crude protein, % 19.11 Net energy, MJ/kg 8.88 SIDLysine g/NE MJ 1.31 SID Lysine, % 1.16 SID Methionine, % 0.35 Neutraldetergent fibre, % 17.93 Calcium, % 0.68 Available phosphorous, % 0.22¹Provided per kg of complete diet; vitamin A, 8255 IU; vitamin D3, 1000IU; vitamin E, 20 IU; vitamin K, 1.5 mg; riboflavin, 7.5 mg; niacin, 30mg; vitamin B12, 25 μg; pyridoxine, 4.5 mg; biotin, 200 μg; folic acid,1 mg; thiamin, 4 mg; choline, 781 mg; copper, 10 mg; iodine, 0.6 mg;iron, 130 mg; manganese, 40 mg; selenium, 0.3 mg; zinc, 130 mg.

A basal diet is formulated to meet the NRC nutrients recommendations forswine (NRC, 1998; Table 16.1). One batch of the basal diet ismanufactured and split into four portions and each portion subsequentlymixed with additives identified in Table 16.2. The 4 treatments areallotted in a completely randomized design to give 8 pens per treatment.Pigs are offered the experimental diets for 42 days during which feedand water are freely available at all times. Body weight and Feed intakeare recorded on a weekly basis and used to calculate feed conversionratio. The growth performance data (BW, ADFI, ADG and FCR) are subjectedto mixed-liner model using the GLM procedures of SAS.

TABLE 16.2 Treatments identification Phytase¹ Diet Treatment ID (FTU/kgof feed) Xylanase Control 1 500 FTU 0 Control + FveXyn4 2 500 FTU 2000U/kg Control + Commercial 3 500 FTU 75 ppm xylanase² ¹Phytase fromDanisco Animal Nutrition ²Econase XT ® from AB Vista FveXyn4 results inbetter body weight gain, specifically 9.9% higher average daily gain(over 42 days) than a commercial xylanase from AB Vista. Pigs exhibiteda better FCR and heavier final body weight when receiving FveXyn4.

16.2 Results and Discussion

FveXyn4 results in better body weight gain, specifically 9.9% higheraverage daily gain (over 42 days) than a commercial xylanase from ABVista. Pigs exhibited a better FCR and heavier final body weight whenreceiving FveXyn4.

TABLE 16.3 Effect of new xylanase on growth performance of growing pigsfed wheat based diets Initial Final Average body body daily Average Feedweight, weight, feed intake, daily gain, conversion kg kg grams/daygrams/day ratio, g/g Control 23.9 61.8 2104 904ab 2.33 FveXyn4 23.0 63.12075 954a 2.18 Commercial 23.2 59.6 2021 868b 2.33 xylanase (Econase ®XT) SEM 0.48 1.16 61.2 23.7 0.07

Example 17

FveXyn4 in Animal Feed—Pigs

17.1 Materials and Methods

The experiment is conducted to evaluate the efficacy of FveXyn4 on ilealnutrients and energy digestibility in growing pigs fed wheat/wheat branbased diets. The protocol for the experiment is reviewed and approved byan Institutional Animal Care and Use Committee and relevant welfareguidelines for the Country are used. Six growing barrows (initial bodyweight of 30 kg) are equipped with a T-cannula in the distal ileum forthe purpose of the experiment. Pigs are of the off springs ofG-Performer boars that were mated to Fertilium 25 females (Genetiporc,Alexandria, Minn., USA) and are housed in individual pens (1.2×1.5 m) inan environmentally controlled room. Each pen is equipped with a feederand a nipple drinker and has fully slatted concrete floors.

TABLE 17.1 Composition of the basal diet Item Level Hard Wheat 56.57Wheat Bran 5.06 Wheat middlings 19.94 Soybean Meal 13.50 Soybean oil0.99 L-Lysine HCl 0.73 DL-Methionine 0.17 L-Threonine 0.30 L-Tryptophan0.01 Digestibility marker (celite) 0.30 Salt 0.47 Limestone 1.38Monocalcium Phosphate 0.08 Vitamins/Trace minerals premix¹ 0.50Calculated provisions Crude protein, % 19.11 Net energy, MJ/kg 8.88 SIDLysine g/NE MJ 1.31 SID Lysine, % 1.16 SID Methionine, % 0.35 Neutraldetergent fibre, % 17.93 Calcium, % 0.68 Available phosphorous, % 0.22¹Provided the following quantities of vitamins and trace minerals perkilogram of complete diet: Vitamin A as retinyl acetate, 11,128 IU;vitamin D₃ as cholecalciferol, 2,204 IU; vitamin E as DL-alphatocopheryl acetate, 66 IU; vitamin K as menadione nicotinamidebisulfite, 1.42 mg; thiamin as thiamine mononitrate, 0.24 mg;riboflavin, 6.58 mg; pyridoxine as pyridoxine hydrochloride, 0.24 mg;vitamin B₁₂,0.03 mg; D-pantothenic acid as D-calcium pantothenate, 23.5mg; niacin as nicotinamide and nicotinic acid, 44 mg; folic acid, 1.58mg; biotin, 0.44 mg; Cu, 10 mg as copper sulfate; Fe, 125 mg as ironsulfate; I, 1.26 mg as potassium iodate; Mn, 60 mg as manganese sulfate;Se, 0.3 mg as sodium selenite; and Zn, 100 mg as zinc oxide.

TABLE 17.2 Treatments identification Phytase¹ Diet Treatment ID (FTU/kgof feed) Xylanase Control 1 500 FTU 0 Control + FveXyn4 2 500 FTU 2000U/kg Control + Commercial 3 500 FTU 75 ppm xylanase² ¹Phytase fromDanisco Animal Nutrition ²Econase XT ® from AB Vista

A basal diet is formulated to meet the NRC nutrients recommendations forswine (Table 17.1). One batch of the basal diet is manufactured andsplit into four portions and each portion subsequently mixed withadditives identified in Table 17.2, The experiment was designed andconducted according to a 4×4 Latin square design with 2 added columns togive 6 replicates per diet. All pigs are fed at a level of 3 times theirmaintenance energy requirement (106 kcal ME per kg^(0.75); NRC, 1998),and provided at 0800 and 1700 h. Animals have free access to waterthrough a bowl-type drinker. Pig weights are recorded at the beginningand at the end of each period and the amount of feed supplied each dayis recorded. Each experimental period lasted for 7 d. The initial 5 daysof each period is considered an adaptation period to the diet. Ilealdigesta are collected for 8 h on d 6 and 7 using standard operatingprocedures. In brief, a plastic bag is attached to the cannula barreland digesta flowing into the bag is collected. Bags are removed wheneverthey are filled with digesta—or at least once every 30 min and areimmediately frozen at −20° C. On the completion of one experimentalperiod, animals are deprived of feed overnight and the followingmorning, a new experimental diet is offered.

At the end of the experiment, ileal samples are thawed, mixed withinanimal and diet, and a sub-sample is collected for chemical analysis. Asample of basal diet is also collected and analyzed. Digesta samples arelyophilized and finely ground prior to chemical analysis. All samplesare analyzed for dry matter, Titanium, gross energy, crude protein, fatand neutral detergent fibre according to standard procedures (AOAC,2005). The values for apparent ileal digestibility of energy andnutrients are calculated as described previously (Stein et al., 2007Livestock Science, 2007 vol. 109, issue 1 part 3 p282-285 and J ANIM SCIJanuary 2007 vol. 85 no. 1 172-180). Data are analyzed using the MIXEDprocedures of SAS.

17.2 Results and Discussion

Results indicate that pigs fed FveXyn4 have significantly higher(P<0.05) apparent ileal digestibility of crude protein and fat thancontrol and the commercial xylanase fed pigs (Table 17.3). Resultssuggest that FveXyn4 is effective in unlocking energy in fibrous feedfed to growing pigs by breaking down fibres. Indeed, pigs fed FveXyn4show increased fibre digestibility by a range of 6 to 11 percentageunits relative to the control and commercial xylanase (Table 17.3).Subsequently pigs fed FveXyn4 extract 106 and 219 kcal extra (Table17.3) energy compared to pigs fed the control and commercial xylanase,respectively.

TABLE 17.3 Effect of new xylanase on apparent ileal nutrients and fibreenergy digestibility (%) and energy utilization (kcal/kg) in growingpigs fed wheat based diets Neutral Crude detergent Dry matter proteinFat fibre Energy Control 68.0ab 74.7b 62.2b 66.3ab 3,168ab FveXyn4 71.2a77.0a 67.7a 72.1a 3,274a Commercial 65.8b 74.6b 56.3b 61.3b 3,055bxylanase (Econase ® XT) SEM  1.44  0.84  2.03  2.65   47.6

Example 18

FveXyn4 In Animal Feed—Pigs

18.1 Materials and Methods

The efficacy of FveXyn4 on ileal and total tract nutrients and energydigestibility in growing pigs fed corn-corn DDGS and wheat/wheat branbased diets is studied. An Animal Care and Use Committee approves theuse of the pigs and pigs are cared for according to the relevant welfareguidelines for the Country. A total of 24 barrows([♀Yorkshire×Landrace]×♂Duroc; initial body weight of 30 kg) areequipped with a T-cannula in the distal ileum for the purpose of theexperiment. The pigs are individually housed in metabolism crates that asmooth transparent plastic sides and plastic-covered expanded metalsheet flooring in a temperature-controlled room (22±2° C.).

TABLE 18.1 Basal diets used Item Corn based Wheat based Corn 41.44 USCorn DDGS 40.00 Hard Wheat 56.57 Wheat Bran 5.06 Wheat middlings 19.94Soybean Meal 15.00 13.50 Tallow 0.99 L-Lysine HCl 0.75 0.73DL-Methionine 0.10 0.17 L-Threonine 0.25 0.30 L-Tryptophan 0.06 0.01Digestibility marker (celite) 0.30 0.30 Salt 0.30 0.47 Limestone 1.301.38 Monocalcium Phosphate 0.00 0.08 Vitamins/Trace minerals premix¹0.50 0.50 Calculated provisions Crude protein, % 21.44 19.11 Net energy,MJ/kg 8.88 8.88 SID Lysine g/NE MJ 1.31 1.31 SID Lysine, % 1.16 1.16 SIDMethionine, % 0.35 0.35 Neutral detergent fibre, % 20.63 17.93 Calcium,% 0.67 0.68 Available phosphorous, % 0.22 0.22 ¹The vitamin and tracemineral premix provided the following (per kg of diet): vitamin A,11,000 IU; vitamin D3, 2,756 IU; vitamin E, 55 IU; vitamin B12, 55 μg;riboflavin, 16,000 mg; panthothenic acid, 44.1 mg; niacin, 82.7 mg; Zn,150 mg; Fe, 175 mg; Mn, 60 mg; Cu, 17.5 mg; I, 2 mg; and Se, 0.3 mg

Respective basal diets are formulated to meet the NRC nutrientrecommendations for swine (NRC, 1998 Table 18.1). In each experiment,one batch of the basal diet is manufactured and split into four portionsand each portion subsequently mixed with additives identified in Table18.2.

TABLE 18.2 Treatments identification Phytase¹ Diet Treatment ID (FTU/kgof feed) Xylanase Control 1 500 FTU 0 Control + FveXyn4 2 500 FTU 4000U/kg Control + Commercial 3 500 FTU 100 ppm xylanase² ¹Phytase fromDanisco Animal Nutrition ²Econase XT ® from AB Vista

The experiment is designed and conducted as two period cross-over designin which ail corn diets are run in period one and all wheat diets run inperiod two. Within a period, the 4 treatments will be allocated to pigsin a completely randomized design to give 6 replicates per treatment.Pigs are fed a common commercial diet for a week before commencement ofthe second period. The pigs are fed their respective diets in two equalportions at 0830 and 1630. Daily feed allowance is based on the pig's BWat the beginning of the period and is calculated to supply 2.6 times theestimated maintenance requirements. Each experimental period lasts for14 d: d 7 for adaptation, d 8 and 9 for grab fecal collection and d 10and 11 for ileal digesta collection to examine coefficient of apparentileal and total tract digestibility of N, DM, energy and crude fat. Pigsare allowed free accessible to water from nipple drinkers located ineach pen at all times. Digesta flow measurements and blood samplescollection are conducted from d 12 to 14. Data id analysed using GLMprocedures of SAS. Statistical significance is accepted at P<0.05.

18.2 Results and Discussion

Preliminary results indicate that pigs fed FveXyn4 have significantlyimproved energy digestibility.

Example 19

FveXyn4 in Animal Feed (Poultry)

19.1 Materials and Methods

An experiment is conducted to evaluate the efficacy of FveXyn4 on growthperformance of broiler chickens fed corn/corn DDGS based diets andwheat/wheat bran based diets. The experimental procedures are approvedby an Animal Ethics Committee and, complied with relevant welfareguidelines for the Country. A two-phase feeding programme (starter andfinisher) is used (Table 1). The starter and finisher diets are offeredfrom d 0 to 21 and 22 to 42, respectively.

TABLE 19.1 Composition of the basal diets¹ Starter, d 0-21 Finisher, d22-42 Corn Wheat Corn Wheat Corn 57.39 — 58.50 — Corn DDGS 11.00 — 15.00— Wheat — 60.17 — 63.30 Wheat Bran — 9.00 — 13.00 Soybean meal, 45%26.50 22.34 19.00 14.00 Tallow 1.75 4.85 3.20 5.95 Vitamin-mineralpremix² 0.33 0.33 0.33 0.33 Sodium bicarbonate 0.20 0.22 0.20 0.29 Salt0.38 0.38 0.34 0.35 Monocalcium phosphate 0.35 0.37 0.13 0.20 Limestone1.700 1.69 1.70 1.65 L-Lysine-HCl 0.135 0.25 0.20 0.34 DL-methionine0.185 0.23 0.13 0.19 L-threonine 0.100 0.19 0.09 0.22 Calculatedprovisions Crude protein 21.0 21.1 18.6 18.2 ME (MJ/kg) 12.5 12.2 12.912.5 Calcium 0.81 0.80 0.75 0.73 Available Phosphorous 0.25 0.25 0.210.21 Sodium 0.23 0.22 0.23 0.22 Digestible Lysine 1.01 0.99 0.90 0.87Digestible Methionine 0.47 0.47 0.39 0.39 Digestible Threonine 0.74 0.740.64 0.66 Digestible Tryptophan 0.19 0.21 0.16 0.17 ¹A commercialphytase from Danisco Animal Nutrition top dressed to supply 500 FTU/kgof final feed ²Supplied per kilogram of diet: antioxidant 100 mg;biotin, 0.2 mg; calcium pantothenate, 12.8 mg; cholecalciferol, 60 μg;cyanocobalamin, 0.017 mg; folic acid, 5.2 mg: menadione, 4 mg; niacin,35 mg; pyridoxine, 10 mg; trans-retinol, 3.33 mg; riboflavin, 12 mg;thiamine, 3.0 mg; dl-α-tocopheryl acetate, 60 mg; choline chloride, 638mg; Co, 0.3 mg; Cu, 3.0 mg; Fe, 25 mg; I, 1 mg; Mn, 125 mg; Mo, 0.5 mg;Se, 200 μg; Zn, 60 mg.

Two basal diets, one based on wheat/wheat bran and soybean meal, and theother based on corn/corn DDGS and soybean meal, are formulated to meetor exceed the recommended requirements for nutrients, except AME, forbroilers (Table 19.1). From each basal diet, four experimental diets aredeveloped to constitute control, FveXyn4, commercial xylanases 1 andcommercial xylanases 2 as identified in Table 19.2.

TABLE 19.2 Treatments identification Phytase¹ Diet Treatment ID (FTU/kgof feed) Xylanase Control 1 500 FTU 0 Control + FveXyn4 2 500 FTU 1250U/kg Control + Commercial 3 500 FTU 50 ppm xylanase² ¹Phytase fromDanisco Animal Nutrition ²Econase XT ® from AB Vista

Male broiler (Ross 308) chicks are obtained as day-olds from acommercial hatchery. The chicks are individually weighed and allocatedto 72 brooder cages (8 chicks per cage) and the 8 dietary treatmentsrandomly assigned to eight cages each. On day 12, the birds aretransferred to grower cages. The space allocation per bird in brooderand grower cages is 530 and 640 cm², respectively. The brooder andgrower cages are housed in environmentally controlled rooms. Thetemperature is maintained at 31° C. in the first week and then graduallyreduced to 22° C. by the end of third week. The birds receive 20 hoursfluorescent illumination and, allowed free access to the diets andwater. Body weights and feed intake are recorded at weekly intervalsthroughout the 42-day experimental period. Mortality is recorded daily.Any bird that dies is weighed and the weight is used to adjust FCR. Feedconversion ratios are calculated by dividing total feed intake by weightgain of live plus dead birds. Data are analysed as a two-way factorialarrangement of treatments using the General Linear Models procedure ofSAS (2004).

19.2 Results and Discussion

Compared with the control and commercial xylanase 2, FveXyn4 Improvesgain and FCR in both corn and wheat based diets (Table 19.3). Thisdemonstrates the efficaciousness of FveXyn4 in mitigating the negativeeffects of the insoluble and soluble fibrous fractions in cerealingredients that may limit poultry performance.

TABLE 19.3 Effect of new xylanase on growth performance of broilerchickens fed corn-corn DDGS and wheat/wheat bran based diets Initialbody Final body Feed Body weight Feed conversion Treatments weight, gweight, g intake, g gain, g ratio, g/g Grain Xylanase Corn Control 38.32151 3738 2113   1.81 Corn FveXyn4 38.3 2306 3946 2268   1.76 CornCommercial 38.5 2304 3969 2265   1.78 xylanase Wheat Control 38.4 24774056 2438   1.72 Wheat FveXyn4 38.3 2678 4294 2639   1.69 WheatCommercial 38.2 2456 4077 2418   1.71 xylanase SEM  0.17  56.5  105 56.5   0.02 Main effects, grains Corn 38.3 2252b 3851b 2214b   1.77aWheat 38.3 2588a 4165a 2550a   1.68b SEM  0.09  28.2  52.5  28.2   0.01Main effects, xylanases Control 38.3 2314b 3847 2275b   1.76a FveXyn438.3 2492a 4120 2453a   1.72b Commercial 38.3 2380b 4023 2342b   1.74abxylanase SEM  0.12  39.9  74.3  39.9   0.01 Probabilities Grain —  <0.01 <0.01  <0.01 <0.01 Xylanase —  <0.01    0.22  <0.01 <0.01 Grain and —   0.04    0.37    0.04   0.05 xylanases interaction Within a column,means with different letters are significantly different, (P < 0.05).

Example 20

FveXyn4 in Animal Feed (Poultry)

20.1 Material and Methods

The experiment is conducted to evaluate the efficacy of FveXyn4 onenergy and nutrient utilization/retention in broiler chickens fedcorn/corn DDGS based diets. An Animal Care and Use Committee approvedail bird handling and collection procedures. Two-hundred fifty-six malebroiler chicks (Ross 708, Aviagen, Huntsville, Ala.) are housed inelectrically heated battery cages (model SB 4 T, Alternative DesignManufacturing, Siloam Springs, Ark.) in an environmentally controlledroom for a 21 day trial. Battery temperatures on d 1 to 8, d 8 to 15,and d 15 to 21 are kept at 35, 32, and 27° C., respectively.

A corn-based basal diet (Table 20.1) is formulated to meet the nutrientrequirements of the broiler chicken. From the basal diet, fourexperimental diets are developed to constitute control, FveXyn4, andcommercial xylanase as identified in Table 20.2.

TABLE 20.1 Composition of the basal diet Ingredients, g/kg Levels Corn554.4 Corn-DDGS 110.0 Soybean Meal 244.4 Soybean Oil 10.0 L-Lysine HCl4.3 DL-Methionine 2.7 L-Threonine 1.1 Sodium Bicarbonate 2.0 Salt 2.2Limestone (A) 15.3 MCP (B) 5.6 Vitamin-Min premix (C) 3.0 TiO2 Premix(D) 25.0 Phytase premix (E) 10.0 Calculated provisions Crude protein,g/kg 211.4 MEP, MJ/kg 115.1 Calcium, g/kg 8.9 Available phosphorous,g/kg 2.8 Digestible Lysine, g/kg 11.5 Digestible Methionine, g/kg 5.5 A.38% Ca B. 16% Ca, 21% P. C. Supplies the following per kg DIET: Vit. A,5484 IU; Vit. D3, 2643 ICU; Vit E, 11 IU; Menadione sodium bisulfite,4.38 mg; Riboflavin, 5.49 mg; d-pantothenic acid, 11 mg; Niacin, 44.1mg; Choline chloride, 771 mg; Vit B12, 13.2 ug; Biotin, 55.2 ug;Thiamine mononitrate, 2.2 mg; Folic acid, 990 ug; Pyridoxinehydrochloride, 3.3 mg; I, 1.11 mg; Mn, 66.06 mg; Cu, 4.44 mg; Fe, 44.1mg; Zn; 44.1 mg; Se, 300 ug. Also contains per g of premix: Vit. A 1828IU; Vit. D3, 881 ICU; Vit E, 3.67 IU; Menadione sodium bisulfite, 1.46mg; Riboflavin, 1.83 mg; d-pantothenic acid, 3.67 mg; Niacin, 14.69 mg;Choline chloride, 257 mg; Vit B12, 4.4 ug; Biotin, 18.4 ug; Thiaminemononitrate, 735 ug; Folic acid, 330 ug, Pyridoxine hydrochloride, 1.1mg; I, 370 ug; Mn, 22.02 mg; Cu, 1.48 mg; Fe, 14.69 mg; Zn, 14.69 mg;Se, 100 ug. D. Prepared as 5 g of TiO2 added to 20 g of fine ground SBM.E. Prepared as 0.2 g of phytase added to 9.8 g of fine ground SBM

TABLE 20.2 Treatments identification Phytase¹ Diet Treatment ID (FTU/kgof feed) Xylanase Control 1 500 FTU 0 Control + FveXyn4 2 500 FTU 1250U/kg Control + Commercial 3 500 FTU 50 ppm xylanase² ¹Phytase fromDanisco Animal Nutrition ²Econase XT ® from AB Vista

Upon arrival at the research facility, chicks are weighed, grouped into4 blocks by blocks, and randomly allocated to 4 dietary groups in eachblock with 8 birds per cage in a randomized complete block design. Thechicks are fed experimental diets (mash) ad libitum from d 1 and arealso allowed ad libitum access to clean drinking water. Excreta arecollected twice daily on d 19, 20, and 21 post-hatch. During collection,waxed paper is placed in trays under the cages and excreta on the waxedpaper are collected. The collected excreta samples are pooled per cageover the 3 d, stored in a freezer, dried, and ground to pass through a0.5-mm screen using a mill grinder (Retsch ZM 100, GmbH & Co. K.C.,Haan, Germany). Excreta and diet samples are analyzed for gross energy,dry matter, fat and neutral detergent fibre using standard procedures(AOAC, 2005) for calculation of apparent retentions and AME. Data weresubsequently subjected to GLM procedures of SAS.

20.2 Results and Discussion

Birds fed FveXyn4 retain more fat and fibre and subsequently extractedmore energy (˜57 kcal/kg) compared with the control fed birds (Table 6).Furthermore, FveXyn4 fed birds have significantly higher fibre retentioncompared to the commercial xylanase.

TABLE 20.3 Effect of new xylanase on apparent nutrients and fibreretention (%) and energy utilization (kcal/kg) in broiler chickens fedcorn-corn DDGS based diets Neutral Crude detergent Dry matter proteinFat fibre Energy Control 67.5ab 60.4 51.6b 27.4b 3107b FveXyn4 68.2ab60.1 57.8a 30.1a 3164a Commercial 68.8a 60.5 52.2ab 26.8b 3156abxylanase (Econase ® XT) SEM  0.48  0.91  2.05  1.15  21.8 Within acolumn, means with different letters are significantly different, (P <0.05).

Example 21

FveXyn4 in Animal Feed (Poultry)

21.1 Material and Methods

An experiment is conducted to evaluate the efficacy of FveXyn4 on energyand nutrient utilization/retention in broiler chickens fed corn/cornDDGS based diets.

The experiment is approved by an Animal Experimentation Committee andconforms to the protocol required by relevant regulations. The birds areallocated to dietary treatments when they are 14 days old. At day old,the birds are brooded together and receive a commercial corn-soybeanmeal pre-experimental diet. The experiment is carried out in anenvironmentally controlled house.

TABLE 21.1 Composition of the basal diet Ingredient, % Level Corn 54.4Corn DDGS 11.0 Soybean Meal 28.9 Soybean Oil 1.00 L-Lysine HCl 0.43DL-Methionine 0.27 L-Threonine 0.11 Sodium Bicarbonate 0.20 Salt 0.22Limestone 1.53 Monocalcium phosphate 0.56 Vitamin mineral premix 1.00Titanium dioxide 0.30 Calculated provisions 100 Crude protein, % 21.1MEP, MJ/kg 11.5 Calcium, % 0.89 Available phosphorous 0.28 Digestiblelysine 1.15 Digestible methionine 0.55 The premix provided (units kg⁻¹diets): Vit A 16,000 iu; Vit D₃ 3,000 iu; Vit E 75 iu; Vit B₁ 3 mg; VitB₂ 10 mg; Vit B₆ 3 mg; Vit B₁₂ 15 μg; Vit K₃ 5 mg; Nicotinic acid 60 mg;Pantothenic acid 14.5 mg; Folic acid 1.5 mg; Biotin 275 μg; Cholinechloride 250 mg; Iron 20 mg; Copper 10 mg; Manganese 100 mg; Cobalt 1mg; Zinc 82 mg; Iodine 1 mg; Selenium 0.2 mg; Molybdenum 0.5 mg.

TABLE 21.2 Treatments identification Phytase¹ Diet Treatment ID (FTU/kgof feed) Xylanase Control 1 500 FTU 0 Control + FveXyn4 2 500 FTU 1250U/kg Control + Commercial 4 500 FTU 50 ppm xylanase² ¹Phytase fromDanisco Animal Nutrition ²Econase XT ® from AB Vista

A corn-based basal diet (Table 21.1) is formulated to meet the nutrientrequirements of the broiler chicken. From the basal diet, fourexperimental diets are developed to constitute control, FveXyn4 andcommercial xylanase as identified in Table 21.2. One-hundred andninety-two 14-d old birds are wing tagged and allocated to 4 treatments,with 8 replicate cages per treatment and 6 birds per replicate cage. Theexperimental design is a randomized complete block with blocks randomlyallocated to spaces within a room. Birds are fed experimental diets fromday 14 to day 21. Water from the mains is provided to the birds adlibitum. The total excreta voided are collected on days 19 to 21. Thisenables calculation of apparent total tract retention using totalcollection. The excreta are dried in forced air oven prior to chemicalanalyses. The diets and excreta are chemically analyzed. Dry matter (DM)is determined by drying in a force draft oven at 100° C. for 24 hours.The crude fat is determined using Soxhlet extractor system (AOAC920.39). Nitrogen content is determined by combustion method using aLeco system (Sweeney, 1998). Titanium is determined using the method ofShort et al, (1996). Gross energy is determined in an adiabaticcalorimeter. Data are subsequently subjected to GLM procedures of SAS.

21.2 Results and Discussion

Compared with the control, FveXyn4 increases (P<0.05) retention of drymatter, crude protein and fat (Table 21.3) in a corn-corn DDGS baseddiet. Furthermore, FveXyn4 has higher retention of dry matter, proteinand fat relative to commercially available feed xylanase. Thisdemonstrates the efficaciousness of FveXyn4 in mitigating the negativeeffects of the insoluble and soluble fibrous fractions in corn grain andcorn-co-products. Indeed, addition of FveXyn4 in the control dietresults in significant energy uplift of 116 kcal/kg.

TABLE 21.3 Effect of new xylanase on apparent nutrients and fibreretention (%) and energy utilization (kcal/kg) in broiler chickens fedcorn-corn DDGS based diets Crude Dry matter protein Fat Energy Control73.5bc 69.2ab 76.1b 3431b FveXyn4 76.1a 72.2a 79.6a 3547a Commercial72.7c 66.2b 78.9ab 3428b xylanase (Econase ® XT) SEM  0.70  1.32  1.12 30.6 Within a column, means with different letters are significantlydifferent, (P < 0.05).

Example 22

FveXyn4 in Animal Feed (Poultry)

22.1 Materials and Methods

An experiment is conducted to evaluate dose response efficacy of FveXyn4on growth performance of broiler chickens fed corn/corn DDGS based dietsand wheat/wheat bran based diets. The experimental procedures areapproved by the Institutional Animal Ethics Committee and, comply withthe relevant welfare guidelines of the Country. A two-phase feedingprogramme (starter and finisher) is used (Table 22.1). The starter andfinisher diets are offered from d 0 to 21 and 22 to 42, respectively.

TABLE 22.1 Composition of the basal diets¹ Starter, d 0-21 Finisher, d22-42 Corn Wheat Corn Wheat Corn 57.39 — 58.50 — Corn DDGS 11.00 — 15.00— Wheat — 60.17 — 63.30 Wheat bran — 9.00 — 13.00 Soybean meal, 45%26.50 22.34 19.00 14.00 Tallow 1.75 4.85 3.20 5.95 Vitamin-mineralpremix² 0.33 0.33 0.33 0.33 Sodium bicarbonate 0.20 0.22 0.20 0.29 Salt0.38 0.38 0.34 0.35 Monocalcium phosphate 0.35 0.37 0.13 0.20 Limestone1.700 1.69 1.70 1.65 L-Lysine-HCl 0.135 0.25 0.20 0.34 DL-methionine0.185 0.23 0.13 0.19 L-threonine 0.100 0.19 0.09 0.22 Calculatedprovisions Crude protein 21.0 21.1 18.6 18.2 ME (MJ/kg) 12.5 12.2 12.912.5 Calcium 0.81 0.80 0.75 0.73 Available Phosphorous 0.25 0.25 0.210.21 Sodium 0.23 0.22 0.23 0.22 Digestible Lysine 1.01 0.99 0.90 0.87Digestible Methionine 0.47 0.47 0.39 0.39 Digestible Threonine 0.74 0.740.64 0.66 Digestible Tryptophan 0.19 0.21 0.16 0.17 ¹Phytase B topdressed to supply 500 FTU/kg of final feed ²Supplied per kilogram ofdiet: antioxidant, 100 mg; biotin, 0.2 mg; calcium pantothenate, 12.8mg; cholecalciferol, 60 μg; cyanocobalamin, 0.017 mg; folic acid, 5.2mg; menadione, 4 mg; niacin, 35 mg; pyridoxine, 10 mg; trans-retinol,3.33 mg; riboflavin, 12 mg; thiamine, 3.0 mg; dl-α-tocopheryl acetate,60 mg; choline chloride, 638 mg; Co, 0.3 mg; Cu, 3.0 mg; Fe, 25 mg; I; 1mg; Mn, 125 mg; Mo, 0.5 mg; Se, 200 μg; Zn, 60 mg.

Two basal diets, one based on wheat/wheat bran and soybean meal, and theother based on corn/corn DDGS and soybean meal, are formulated to meetor exceed the recommended requirements for nutrients, except AME, forbroilers (Table 22.1). From each basal diet, three experimental dietsare developed to constitute control, 3 doses of FveXyn4 and commercialxylanase as identified in Table 22.2.

TABLE 22.2 Treatments identification Treatment Phytase Diet ID (FTU/kgof feed) Xylanase Control 1 500 FTU 0 Control + FveXyn4-dose 1 2 500 FTU1250 U/kg Control + FveXyn4-dose 2 3 500 FTU 2500 U/kg Control +FveXyn4-dose 3 4 500 FTU 5000 U/kg Control + Commercial 5 500 FTU 100ppm xylanase¹ ¹AB Vista

Male broiler (Ross 308); chicks are obtained as day-olds from acommercial hatchery. The chicks are individually weighed and allocatedto 60 brooder cages (8 chicks per cage) and the 10 dietary treatmentsrandomly assigned to six cages each. On day 12, the birds aretransferred to grower cages. The space allocation per bird in brooderand grower cages is 530 and 640 cm², respectively. The brooder andgrower cages are housed in environmentally controlled rooms. Thetemperature is maintained at 31° C. in the first week and then graduallyreduced to 22° C. by the end of third week. The birds receive 20 hoursfluorescent illumination and, allowed free access to the diets andwater. Body weights and feed intake are recorded at weekly intervalsthroughout the 42-day experimental period. Mortality is recorded daily.Any bird that die is weighed and the weight is used to adjust FCR. Feedconversion ratios are calculated by dividing total feed intake by weightgain of live plus dead birds. Data are analysed as a two-way factorialarrangement of treatments using the General Linear Models procedure ofSAS (2004).

22.2 Results and Discussion

Compared with the control and commercial xylanase, FveXyn4 improves feedconversion efficiency in a dose dependent manner in both corn and wheatbased diets (Table 22.3). These observations suggested more value of thexylanase is derived at higher inclusion rate. This demonstrates theefficaciousness of FveXyn4 in mitigating the negative effects of theinsoluble and soluble fibrous fractions in cereal ingredients that maylimit poultry ability to utilize dietary nutrients to support growthperformance.

TABLE 22.3 Dose response effect of new xylanase on growth performance ofbroiler chickens fed corn-corn DDGS and wheat/wheat bran based dietsInitial Final Feed Body Feed body body intake, weight conversionTreatments weight, g weight, g g grain, g ratio, g/g Grain Xylanase CornControl 33.50 2569 4162 2536 1.726 Corn FveXyn4-Dose1 33.54 2584 43352551 1.708 Corn FveXyn4-dose2 33.65 2585 4224 2552 1.671 CornFveXyn4-Dose3 33.54 2580 4177 2546 1.683 Corn Commercial 33.98 2646 42952612 1.677 xylanase 2 Wheat Control 33.56 2508 4456 2474 1.824 WheatFveXyn4-Dose1 33.98 2485 4285 2451 1.814 Wheat FveXyn4-dose2 33.63 26744413 2641 1.656 Wheat FveXyn4-Dose3 33.81 2707 4330 2673 1.629 WheatCommercial 34.13 2590 4297 2556 1.694 xylanase 2 SEM 0.22 55.2 56.6 91.356.5 Main effects, grains Corn 33.64 2593 4238b 2559 1.693 Wheat 33.822593 4356a 2559 1.723 SEM 0.10 25.30 40.82 25.28 0.018 Main effects,xylanases Control 33.53 2539 4309 2505 1.775a FveXyn4-Dose1 33.76 25354310 2501 1.761ab FveXyn4-Dose2 33.64 2630 4318 2596 1.664cFveXyn4-dose3 33.68 2643 4254 2610 1.656c Commercial Xylanase 2 34.052618 4296 2584 1.686bc SEM 0.152 40.00 64.55 39.98 0.028 ProbabilitiesGrain — 0.993 0.046 0.989 0.237 Xylanase — 0.165 0.958 0.166 0.008 Grainand Xylanase — 0.190 0.329 0.189 0.212 interaction Within a column,means with different letters are significantly different, (P < 0.05)

Example 23

FveXyn4 in Combination with Protease

The effect of FveXyn4 in combination with protease was investigated onthe solubilization of pentosan and protein from prepared insoluble DDGS.

23.1 Materials and Methods

Enzyme Samples

The xylanase used in this study is a new GH10 xylanase from Fusariumverticilloides (designated FveXyn4) expressed in Trichoderma reesei,wherein the xylanase was used in purified form—this enzyme may bereferred to herein as FveXyn4.

The protease used in this study is the Muitifect P-3000 product(available from Danisco Animal Nutrition). The preparation of proteasewas performed just prior to loadings. Proper amount of stock solutionwas diluted in cooled MQ-water and mixed while kept on ice. One proteaseunit (U) was defined as the release of 1.0 μg of phenolic compound(expressed as tyrosine equivalents) from a casein substrate per minute.

Substrate Preparation

For the preparation of insoluble DDGS substrate, removal of solublenon-starch polysaccharides (S-NSP) was performed according to BachKnudsen (Bach Knudsen, K. E., Carbohydrate and lignin contents of plantmaterials used in animal feeding. Animal Feed Science and Technology1997, 67, 319-338.); Milled DDGS (<212 μm) and acetate/CaCl₂-buffer (0,1 M/20 mM, pH 5.0) was added together with thermostable α-amylase(E-BLAAM 53.7 U/mg, Megazyme International) and incubated for 1 h at100° C. with frequent mixing. Complete degradation of starch was done byincubation with amyloglucosidase (E-AMGDF 36 U/mg, MegazymeInternational) for 2 h at 60° C. After removal of the starch, the S-NSPwas extracted by a phosphate buffer (0.2 M, pH 7.0) and placed at 100°C. for 1 h, followed by centrifugation. The pellet was then thoroughlywashed with phosphate buffer, ethanol (85% v/v), and finally acetone,with centrifugation and discard of supernatant in between washes. Thesample was placed at room temperature until completely dry.

Procedure

FveXyn4 alone or in combination with protease was investigated on thesolubilization of pentosan and protein of prepared insoluble DDGS. 87.5mg of the prepared insoluble DDGS substrate was weighed into 1.5 mleppendorf tubes and mixed with citrate buffer (25 mM, pH 6), xylanase(217 mg/kg substrate and 206 mg/kg substrate for corn—and wheat DDGS,respectively), and protease (8.6×10⁵ U/kg substrate) to a final reactionvolume of 1.0 ml. The incubations were carried out at 4 h, 39° C. and1300 rpm by use of Eppendorf ThermoMixer incubator (Eppendorf). Afterincubation, samples were filtered and analyzed for soluble pentosan and-protein content, as described below. Reactions were performed induplicates.

Protein Quantification

Soluble protein was quantified using the BCA (bicinchoninic acid)Protein Assay Kit from Pierce. The samples were prepared in microtiterplates (25 μl/well) and incubated with 200 μl premixed assay reagent for30 minutes at 37° C., 1100 rpm. The absorbance was measuredspectrophotometrically at 562 nm against a 0-2000 μg/ml Bovine SerumAlbumin (BSA) standard, as described in the manual. Values werecorrected for the amount of added enzymes.

Quantification of C5 Sugars (Pentosans)

The total amount of pentoses brought into solution was measured usingthe method of Rouau and Surget (1994, A rapid semi-automated method ofthe determination of total and water-extractable pentosan in wheatflours. Carbohydrate Polymers, 24, 123-32) with a continuous flowinjection apparatus (FIG. 7). The supernatants were treated with acid tohydrolyse polysaccharides to monosugars. Phloroglucinol (1, 3,5-trihydroxybenzen) was added for reaction with monopentoses andmonohexoses, which forms a coloured complex.

By measuring the difference in absorbance at 550 nm compared to 510 nm,the amount of pentoses in the solution was calculated using a standardcurve. Unlike the pentose-phloroglucinol complex, the absorbance of thehexose-phloroglucinol complex is constant at these wavelengths. Glucosewas added to the phloroglucinol solution to create a constant glucosesignal and further ensure no interference from hexose sugars.

Statistical Analysis

A one-way ANOVA was applied on the experimental data for comparison oftreatments on both the solubilization of pentosan and protein, withpairwise comparisons performed by Holm-Sidak method, using SigmaPlot12.0 (SyStat Software Inc.). Overall significance level at P=0.05.

23.2 Results and Discussion

Pentosan and protein solubilization was measured by incubation ofinsoluble corn and wheat DDGS with xylanase and protease alone and incombination.

The results are shown in FIG. 27 (insoluble corn DDGS) and in FIG. 28(insoluble wheat DDGS).

FIG. 27 shows the effect of the xylanase and protease treatments aloneand in combination on the solubilization of pentosan and protein frominsoluble corn DDGS. Letters a-d are significant different according toon-way ANOVA and Holm-Sidak comparisons with overall significance levelat P=0.05. Error bars indicate S.D.

FIG. 28 shows the effect of the xylanase and protease treatments aloneand in combination on the solubilization of pentosan and protein fromInsoluble wheat DDGS. Letters a-d are significant different according toon-way ANOVA and Holm-Sidak comparisons with overall significance levelat P=0.05. Error bars indicate S.D.

When compared to the effects of xylanase treatment by itself, thecombination of xylanase and protease further increased thesolubilization of protein from both corn and wheat DDGS. Moreinterestingly, addition of protease also significantly increased thesolubilization of pentosan from both corn and wheat DDGS, indicating asynergistic effect where addition of protease increase the accessibilityof the xylanase towards the substrate by opening up the feed matrixstructure through protein degradation. Furthermore, xylanase by itselfand in combination with protease also increase the solubilization ofprotein as compared to control and protease alone, respectively. Thisfurther supports, the theory of a synergistic effect between xylanaseand protease.

Example 24

Comparison of FveXyn4 and FoxXyn2 in Pentosan Solubilisation (Breakdownor Solubilisation of Insoluble Arabinoxylan (AXinsol))

The ability to solubilize insoluble arabinoxylan from corn DDGS was usedas one of the key selection criteria and in this experiment the abilityto solubilise arabinoxylan from corn DDGS is compared for the twoxylanases of the present invention (FyeXyn4 and FoxXyn2).

The two xylanases showed strong and equal performance on pentosansolubilisation from corn DDGS, indicating equal performance of thesehomologous xylanases on solubilisation of arabinoxylan from fibrousby-products.

24.1 Materials and Methods

Enzyme Samples

The xylanases used in this study are:

A GH10 xylanase from Fusarium verticilloides (designated FveXyn4)expressed in Trichoderma reesei, wherein the xylanase was used inpurified form—this enzyme may be referred to herein as FveXyn4, and aGH10 xylanase from Fusarium oxysporum (designated FoxXyn2) expressed inTrichoderma reesei, wherein the xylanase was used in purified form—thisenzyme may be referred to herein as FoxXyn2.

Feed Raw Materials

The feed used in this experiments is corn DDGS.

Pentosan Solubilisation (AXinsol Solubilisation)

The method used for pentosan solubilisation was: 100 mg of feed rawmaterial was transferred to a 2 ml Eppendorf centrifuge tube and theprecise weight recorded. 750 μL incubation buffer (200 mM HEPES, 100 mMNaCl, 2 mM CaCl, pH 6.0) and 900 μl chloramphenicol solution (40 μg/mlin incubation buffer) was added. Enzyme of choice was added to make atotal volume of 1.8 mL.

Each sample was assayed in doublets and in parallel with a blank(incubation without exogenously added enzyme). The samples wereincubated on an Eppendorf thermomixer at 40° C. with shaking. After 2 or18 hours of incubation the supernatant was filtered using 96 wellsfilterplates (Pall Corporation, AcroPrep 96 Filter Plate, 1.0 μm Glass,NTRL, 1 mL well). After filtration the samples were stored at 4° C.until analysis for total amount of C5 sugars, arabinose and xylose.

Quantification of C5 Sugars (Pentosans)

The total amount of pentoses brought into solution was measured usingthe method of Rouau and Surget (1994, A rapid semi-automated method ofthe determination of total and water-extractable pentosan in wheatflours. Carbohydrate Polymers, 24, 123-32) with a continuous flowinjection apparatus (FIG. 7). The supernatants were treated with acid tohydrolyse polysaccharides to monosugars. Phloroglucinol (1, 3,5-trihydroxybenzen) was added for reaction with monopentoses andmonohexoses, which forms a coloured complex. By measuring the differencein absorbance at 550 nm compared to 510 nm, the amount of pentoses inthe solution was calculated using a standard curve. Unlike thepentose-phloroglucinol complex, the absorbance of thehexose-phloroglucinol complex is constant at these wavelengths. Glucosewas added to the phloroglucinol solution to create a constant glucosesignal and further ensure no interference from hexose sugars.

24.2 Result and Discussion

Pentosan solubilisation was monitored in a dose response setup usingfibrous by-product of corn (namely cDDGS).

The results comparing the two xylanases of the present Invention(FveXyn4 and FoxXyn2) are shown in FIG. 29 (in corn DDGS).

FIG. 29 shows solubilisation of pentosans from cDDGS as a function ofxylanase dosage. The xylanases used were the xylanases of the presentinvention (FveXyn4 and FoxXyn2).

Both xylanases performs well on corn and are equally good a breakingdown AXinsol (e.g. solubilising pentosans) in corn based substrateswhich clearly indicates that these two homologous xylanases performequal on solubilisation of arabinoxylan from fibrous by-products.

All publications mentioned in the above specification are hereinincorporated by reference. Various modifications and variations of thedescribed methods and system of the present invention will be apparentto those skilled in the art without departing from the scope and spiritof the present invention. Although the present invention has beendescribed in connection with specific preferred embodiments, it shouldbe understood that the invention as claimed should not be unduly limitedto such specific embodiments. Indeed, various modifications of thedescribed modes for carrying out the invention which are obvious tothose skilled in biochemistry and biotechnology or related fields areintended to be within the scope of the following claims.

1-18. (canceled)
 19. A feed additive composition or a premix comprisinga polypeptide having xylanase activity having at least 85% sequenceidentity with the polypeptide sequence in SEQ ID NO:3, and optionally atleast one mineral and/or at least one vitamin.
 20. The feed additivecomposition or premix according to claim 19 which further comprises oneor more of the enzymes selected from the group consisting of a protease,and a phytase.
 21. (canceled)
 22. A feedstuff comprising a feed additivecomposition or premix according to claim 19 obtained by admixing a feedcomponent with the polypeptide of claim
 19. 23-26. (canceled)
 27. Amethod for degrading xylan in xylan-containing material, by admixingsaid xylan-containing material with a xylanase comprising a polypeptidesequence having at least 85% sequence identity with the polypeptidesequence in SEQ ID NO:3
 28. The method according to claim 27 wherein thearabinoxylan is insoluble arabinoxylan (AXinsol).
 29. The methodaccording to claim 27 wherein the xylan-containing material is selectedfrom one or more of the group consisting of: a feed or feedstuff; a feedcomponent; a grain-based material; a mash; a wort; a malt; maltedbarely; an adjunct, a barley mash; and a cereal flour.
 30. The methodaccording to claim 27 wherein the arabinoxylans are solubilized withoutincreasing viscosity in the reaction medium.
 31. The method according toclaim 29 wherein the feed or feedstuff or feed component comprises orconsists of corn, DDGS (such as cDDGS), wheat, wheat bran or acombination thereof.
 32. The method according to claim 31 wherein thefeed or feedstuff is a corn-based feedstuff.
 33. The method according toclaim 27 wherein the xylanase is used in combination with one or more ofthe enzymes selected from the group consisting of a protease, anamylase, and a phytase.